Mametabolites ekudzivirira muviri ndiwo chinhu chikuru che microenvironment yetumor (TME), asi kunze kwezvishoma, kuzivikanwa kwawo hakusati kwazivikanwa zvakanyanya. Pano, takaongorora matumor nemasero eT kubva mumatumor neascites evarwere vane high-grade serous carcinoma (HGSC) kuti tiratidze metabolome yezvikamu zvakasiyana zveTME. MaAscites nemasero etumor ane musiyano wakakura wemetabolite. Kana tichienzanisa neascites, masero eT anopinza tumor anowedzera zvakanyanya mu 1-methylnicotinamide (MNA). Kunyangwe huwandu hweMNA mumasero eT hwakakwira, kuratidzwa kwe nicotinamide N-methyltransferase (enzyme inokonzeresa kutamiswa kwemapoka emethyl kubva kuS-adenosylmethionine kuenda ku nicotinamide) kunogumira ku fibroblasts nemasero etumor. Pakushanda, MNA inokurudzira masero eT kuti abudise cytokine tumor necrosis factor alpha inokurudzira tumor. Nokudaro, TME-derived MNA inobatsira mukutonga kwemasero eT uye inomiririra chinangwa che immunotherapy chekurapa kenza yevanhu.
Mametabolites anobva mugomarara anogona kuva nemhedzisiro yakakura pakudzivirira immune system kurwisa gomarara, uye humbowo hwakawanda hunoratidza kuti anogonawo kushanda sesimba guru rekufambisa chirwere (1). Kuwedzera kuWarburg effect, basa razvino ratanga kuratidza mamiriro emetabolites emagomarara uye hukama hwayo nemamiriro ekudzivirira etumor microenvironment (TME). Zvidzidzo pamhando dzembeva nemasero eT evanhu zvakaratidza kuti glutamine metabolism (2), oxidative metabolism (3) uye glucose metabolism (4) zvinogona kushanda zvakazvimiririra pamapoka akasiyana-siyana emasero ekudzivirira immune. Mametabolites akati wandei munzira idzi anodzivirira basa remaT cells kurwisa gomarara. Zvave zvichiratidzwa kuti kuvharirwa kwecoenzyme tetrahydrobiopterin (BH4) kunogona kukanganisa kuwanda kwemasero eT, uye kuwedzera kweBH4 mumuviri kunogona kuwedzera mhinduro yekudzivirira immune system inokonzereswa neCD4 neCD8. Pamusoro pezvo, mhedzisiro yekudzivirira immune system yekynurenine inogona kununurwa nekupa BH4 (5). Mu isocitrate dehydrogenase (IDH) mutant glioblastoma, kuburitswa kwe enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) kunodzivisa kushanda kwemasero eT, kuwanda kwawo uye cytolysis Activity (6). Munguva pfupi yapfuura, zvakaratidzwa kuti methylglyoxal, chinhu chinobva muglycolysis, inogadzirwa nemasero ekudzvinyirira anobva ku myeloid, uye kutamiswa kwemasero eT kwemethylglyoxal kunogona kudzivirira mashandiro emasero eT. Mukurapa, kusashanda kwemethylglyoxal kunogona kukunda mashandiro emasero ekudzvinyirira anobva ku myeloid (MDSC) uye kuwedzera kurapa kwekudzivirira kwecheckpoint mumamodeli embeva (7). Zvidzidzo izvi pamwe chete zvinosimbisa basa guru remametabolites anobva kuTME mukudzora mashandiro emasero eT.
Kusashanda zvakanaka kwemasero eT kwave kuchitaurwa zvakanyanya mukenza yemuchibereko (8). Izvi zvinokonzerwa nehunhu hwemetabolism hunowanikwa mukushaya hope uye bundu risingawanzoitiki muropa (9), izvo zvinoguma nekushandurwa kweglucose netryptophan kuita zvigadzirwa zvakaita selactic acid nekynurenine. Lactate yakawanda yekunze kweropa inoderedza kugadzirwa kweinterferon-γ (IFN-γ) uye inoita kuti mapoka e myelosuppressive awedzere (10, 11). Kudya tryptophan kunodzivirira zvakananga kuwanda kwemasero eT uye kunodzivisa T cell receptor signaling (12-14). Pasinei nekucherechedza uku, basa rakawanda rakatenderedza metabolism yemuviri rakaitwa mu in vitro T cell culture uchishandisa optimized media, kana kungogumira kune homologous mouse models in vivo, iyo hapana kana imwe inoratidza zvizere kusiyana kwekenza dzevanhu uye Physiological macro uye micro environment.
Chinhu chinowanzoonekwa nekenza yemuchibereko kupararira kwemasero emudumbu uye kuonekwa kwemasero emudumbu. Kuunganidzwa kwemvura mumasero emudumbu mumasero emudumbu kunobatanidzwa nechirwere chakanyanya uye kufambira mberi kwechirwere (15). Sekureva kwemishumo, chikamu ichi chakasiyana hachina oxygen, chine huwandu hwakawanda hwe vascular endothelial growth factor (VEGF) uye indoleamine 2,3-dioxygenase (IDO), uye chinopinzwa nemasero eT regulatory uye masero e myeloid inhibitory (15-18). Nzvimbo ye metabolic yemasero emudumbu inogona kusiyana neiyo yebundu pacharo, saka kurongwazve kwemasero eT munzvimbo yedumbu hakuna kujeka. Pamusoro pezvo, misiyano mikuru uye kusiyana pakati pemasero emudumbu nemasero emudumbu ari munzvimbo yebundu zvinogona kukanganisa kupinda kwemasero ekudzivirira muviri uye basa rawo pamabundu, uye kumwe kutsvagurudza kunodiwa.
Kuti tigadzirise matambudziko aya, takagadzira nzira yekuparadzanisa masero inonzwisisika uye liquid chromatography tandem mass spectrometry (LC-MS/MS) yekudzidza marudzi akasiyana emasero (kusanganisira masero eCD4 + neCD8 + T) pamwe chete nemukati nepakati pematumbu. Mametabolites ayo anotenderera masero munzvimbo imwechete yeascites nenzvimbo yetumbu yemurwere. Tinoshandisa nzira iyi pamwe chete ne high-dimensional flow cytometry uye single-cell RNA sequencing (scRNA-seq) kuti tipe mufananidzo wakanyatsogadziriswa wemamiriro emetabolism evanhu ava vakakosha. Nzira iyi yakaratidza kuwedzera kukuru kwehuwandu hwe1-methylnicotinamide (MNA) mumasero etumbu T, uye kuyedza kwe in vitro kwakaratidza kuti mhedzisiro yeMNA paT cell function yaisazivikanwa kare. Kazhinji, nzira iyi inoratidza kudyidzana kwemetabolism pakati pematumbu nemasero ekudzivirira muviri, uye inopa ruzivo rwakasiyana mu immune regulation metabolites, izvo zvinogona kubatsira pakurapa T cell-based ovarian cancer immunotherapy mikana yekurapa.
Takashandisa high-dimensional flow cytometry kuti tiverenge panguva imwe chete kutorwa kweglucose [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) uye mitochondrial activity [MitoTracker Deep Red (MT DR)] (7, 19, 20) zviratidzo zvinosiyanisa masero ekudzivirira muviri nemasero egomarara (Table S2 neFigure S1A). Ongororo iyi yakaratidza kuti kana tichienzanisa nemasero eT, ascites nemasero egomarara ane huwandu hwakakwira hweglucose uptake, asi ane mutsauko mudiki mukushanda kwemitochondrial. Avhareji yeglucose uptake yemasero egomarara [CD45-EpCAM (EpCAM)+] inopetwa katatu kusvika kana kana kupfuura yemasero eT, uye avhareji yeglucose uptake yemasero eCD4 + T inopetwa ka1.2 kupfuura yemasero eCD8 + T, zvinoratidza kuti malymphocyte egomarara (TIL) ane zvinodiwa zvakasiyana zve metabolic kunyangwe muTME imwe chete (Figure 1A). Kusiyana neizvi, mashandiro emumitochondrial mumasero egomarara akafanana neemasero eCD4 + T, uye mashandiro emumitochondrial emarudzi ese emasero akakwira kupfuura emasero eCD8 + T (Mufananidzo 1B). Kazhinji, mhedzisiro iyi inoratidza huwandu hwemetabolism. Mabasa emetabolism emasero egomarara akakwira kupfuura emasero eCD4 + T, uye mashandiro emetabolism emasero eCD4 + T akakwira kupfuura emasero eCD8 + T. Pasinei nemhedzisiro iyi mumarudzi esero, hapana mutsauko unogara uripo muchimiro chemetabolism yemasero eCD4 + neCD8 + T kana huwandu hwawo muascites zvichienzaniswa nemagomarara (Mufananidzo 1C). Kusiyana neizvi, muchikamu chemasero eCD45, huwandu hwemasero eEpCAM + mugomarara hwakawedzera zvichienzaniswa neascites (Mufananidzo 1D). Takaonawo musiyano wakajeka wemetabolism pakati pezvikamu zvemasero eEpCAM+ neEpCAM. Masero eEpCAM+ (tumor) ane glucose yakakwira uye mitochondrial kupfuura masero eEpCAM, ayo akakwira zvikuru kupfuura mashandiro emetabolism efibroblasts mumasero egomarara muTME (Mufananidzo 1, E naF).
(A naB) Kupenya kwepakati kweglucose (MFI) (2-NBDG) (A) uye mitochondrial activity yeCD4 + T cells (MitoTracker dema dzvuku) (B) Magirafu anomiririra (kuruboshwe) uye data rakarongwa (Kurudyi), maCD8 + T cells uye maEpCAM + CD45-tumor cells kubva kuascites nebundu. (C) Chiyero cheCD4 + neCD8 + cells (dzemaCD3 + T cells) muascites nebundu. (D) Chikamu cheEpCAM + tumor cells muascites nebundu (CD45−). (E naF) EpCAM + CD45-tumor uye EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) uye mitochondrial activity (MitoTracker dema dzvuku) (F) magirafu anomiririra (kuruboshwe) uye data rakarongwa (Kurudyi) MaAscites nemasero ebundu. (G) Magirafu anomiririra eCD25, CD137 uye PD1 expression ne flow cytometry. (H naI) Kuratidzwa kweCD25, CD137 uye PD1 pamasero eCD4 + T (H) uye masero eCD8 + T (I). (J naK) Naive, central memory (Tcm), effector (Teff) uye effector memory (Tem) phenotypes zvichibva pakutaura kweCCR7 neCD45RO. Mifananidzo inomiririra (kuruboshwe) uye data retabhu (kurudyi) remasero eCD4 + T (J) uye CD8 + T (K) mu ascites uye bundu. P values ​​​​inosarudzwa ne paired t-test (*P<0.05, **P<0.01 uye ***P<0.001). Mutsetse unomiririra varwere vakaenzana (n = 6). FMO, fluorescence minus one; MFI, median fluorescence intensity.
Ongororo yakawedzerwa yakaratidza mimwe misiyano yakakosha pakati pemamiriro eT cell ane resolved highly resolved phenotypic. Iyo activated (Mufananidzo 1, G kusvika I) uye effector memory (Mufananidzo 1, J na K) mumabundu anowanzoitika kupfuura ascites (chikamu cheCD3 + T cells). Saizvozvowo, kuongorora phenotype nekutaura kwe activation markers (CD25 naCD137) uye depletion markers [programmed cell death protein 1 (PD1)] yakaratidza kuti kunyangwe hunhu hwe metabolic hwevanhu ava hwakasiyana (Mufananidzo S1, B kusvika E), Asi hapana musiyano wakakosha we metabolic wakaonekwa nguva dzose pakati pe naive, effector kana memory subsets (Mufananidzo S1, F kusvika I). ​​Mhedzisiro iyi yakasimbiswa nekushandisa machine learning methods kuti ipe otomatiki ma cell phenotypes (21), izvo zvakaratidzawo kuvapo kwenhamba huru ye bone marrow cells (CD45 + / CD3- / CD4 + / CD45RO +) mu ascites yemurwere (Mufananidzo S2A). Pakati pemarudzi ese emasero akaonekwa, huwandu hwemasero aya emyeloid hwakaratidza kutorwa kweglucose kwakanyanya uye kushanda kwemitochondrial (Mufananidzo S2, B kusvika G). Izvi zvinoratidza musiyano wakasimba wemetabolism pakati pemarudzi akawanda emasero anowanikwa muascites nemabundu muvarwere vane HGSC.
Dambudziko guru pakunzwisisa hunhu hweTIL hwemetabonomic ndiko kudiwa kwekubvisa samples dzeT cell dzakachena zvakakwana, mhando uye huwandu kubva kumabundu. Ongororo dzichangobva kuitwa dzakaratidza kuti nzira dzekuronga nekuwedzera beads zvichibva paflow cytometry dzinogona kutungamira kuchinjo mu cellular metabolites profiles (22-24). Kuti tikunde dambudziko iri, takagadzirisa nzira yekuwedzera beads kuti tibvise uye tibvise TIL kubva kukenza ye ovarian yakabviswa nekuvhiyiwa isati yaongororwa neLC-MS/MS (ona Zvinhu neNzira; Mufananidzo 2A). Kuti tiongorore kukanganisa kwese kweprotocol iyi pakuchinja kwemetabolite, takaenzanisa metabolites profiles dzeT cells dzakashandiswa nevanopa hutano mushure medanho rekuparadzanisa bead riri pamusoro apa nemasero asina kuparadzaniswa beads asi akaramba ari pachando. Kuongorora uku kwehunhu kwakawana kuti pane hukama hwakakura pakati pemamiriro maviri aya (r = 0.77), uye technical repeatableability yeboka re86 metabolites ine high repeatableability (Mufananidzo 2B). Nokudaro, nzira idzi dzinogona kuita ongororo chaiyo yemetabolite mumasero ari kusimudzirwa kwemhando yemasero, nokudaro zvichipa puratifomu yekutanga yepamusoro-soro yekuona metabolite chaiyo muHGSC, nokudaro zvichiita kuti vanhu vanzwisise zvakadzama nezve specificity yemasero. Chirongwa chemetabolism yepabonde.
(A) Dhiyagiramu yekuongorora magnetic bead. LC-MS/MS isati yaongororwa, masero achaitwa katatu akatevedzana ekuwedzera magnetic bead kana kuti acharamba ari pachando. (B) Mhedzisiro yerudzi rwekuwedzera pahuwandu hwemetabolites. Avhareji yezviyero zvitatu zvemhando yega yega yekuwedzera ± SE. Mutsetse wegrey unomiririra hukama hwe1:1. Inter-class correlation (ICC) yezviyero zvinodzokororwa zvinoratidzwa mu axis label. NAD, nicotinamide adenine dinucleotide. (C) Dhiyagiramu yekuongorora mafambiro ekuongorora metabolite yemurwere. Ascites kana tumors zvinounganidzwa kubva kuvarwere uye zvinochengetwa. Chikamu chidiki chemuenzaniso wega wega chakaongororwa ne flow cytometry, nepo sampuli dzasara dzakaitwa katatu ekuwedzera masero eCD4+, CD8+ uye CD45. Aya ma fraction emasero akaongororwa uchishandisa LC-MS/MS. (D) Mepu yekupisa yehuwandu hwemetabolite hwakajairwa. Dendrogram inomiririra Ward's clustering yeEuclidean distances pakati pesampuli. (E) Kuongororwa kwechikamu chikuru (PCA) chemuenzaniso wemepu yemetabolism, ichiratidza makopi matatu emuenzaniso wega wega, sampuli kubva kumurwere mumwe chete dzakabatana nemutsetse. (F) PCA yeprofile yemetabolism yemuenzaniso inoenderana nemurwere (kureva, uchishandisa chikamu chekudzokororwa); mhando yemuenzaniso inogumira neganda rakatenderedza. PC1, chikamu chikuru 1; PC2, chikamu chikuru 2.
Tevere, takashandisa nzira iyi yekuwedzera masero kuti tiongorore ma metabolites 99 muCD4+, CD8+ uye CD45-cell fractions mu ascites yekutanga uye bundu revarwere vatanhatu veHGSC (Mufananidzo 2C, Mufananidzo S3A uye Tafura S3 uye S4). Huwandu hwevanhu vanofarirwa hunosvika 2% kusvika 70% yemuenzaniso mukuru wekutanga wemasero mapenyu, uye huwandu hwemasero hunosiyana zvikuru pakati pevarwere. Mushure mekuparadzanisa mabhesi, chikamu chakapfuma chemubereko (CD4+, CD8+ kana CD45-) chinodarika 85% yemasero ese mapenyu mumuenzaniso paavhareji. Nzira iyi yekuwedzera masero inotibvumira kuongorora huwandu hwemasero kubva mu metabolism ye tumor tissue yevanhu, izvo zvisingaite kubva kumasampuli makuru. Tichishandisa iyi protocol, takaona kuti l-kynurenine ne adenosine, aya ma metabolites maviri ane hunhu hwakanaka ekudzivirira immune akakwidziridzwa mumasero e tumor T kana masero e tumor (Mufananidzo S3, B na C). Nokudaro, mhedzisiro iyi inoratidza kuvimbika uye kugona kwetekinoroji yedu yekuparadzanisa masero uye mass spectrometry yekuwana ma metabolites akakosha mumasero evarwere.
Ongororo yedu yakaratidzawo kupatsanurwa kwakasimba kwemasero mukati uye pakati pevarwere (Mufananidzo 2D neMufananidzo S4A). Zvikuru, zvichienzaniswa nevamwe varwere, murwere 70 akaratidza hunhu hwakasiyana hwemasero (Mufananidzo 2E neMufananidzo S4B), zvichiratidza kuti panogona kunge paine kusiyana kukuru kwemasero pakati pevarwere. Zvakakosha kuziva kuti kana tichienzanisa nevamwe varwere (1.2 kusvika 2 litres; Tafura S1), huwandu hwese hwemasero akaunganidzwa mumurwere 70 (80 ml) hwaive hudiki. Kudzora kusiyana kwemasero pakati pevarwere panguva yekuongorora zvikamu zvikuru (semuenzaniso, uchishandisa ongororo yechikamu chekudzokororwa) kunoratidza shanduko dzinogara dziripo pakati pemarudzi emasero, uye marudzi emasero uye/kana microenvironment zvakajeka zvakabatanidzwa zvichienderana neprofile yemetabolite (Mufananidzo 2F). Ongororo yemetabolite imwe chete yakasimbisa mhedzisiro iyi uye yakaratidza mutsauko mukuru pakati pemarudzi emasero nenzvimbo diki. Zvakakosha kuziva kuti mutsauko wakanyanya kuonekwa iMNA, iyo inowanzo kupfuma mumasero eCD45 uye mumasero eCD4+ neCD8+ anopinda mubundu (Mufananidzo 3A). Kune masero eCD4 +, mhedzisiro iyi inonyanya kuoneka, uye MNA mumasero eCD8 + inoita kunge yakakanganiswa zvakanyanya nemamiriro ekunze. Zvisinei, izvi hazvina kukosha, nekuti vatatu chete pavarwere vatanhatu vanogona kuongororwa kuti vane maCD8+ bundu. Pamusoro peMNA, mumhando dzakasiyana dzemasero mu ascites nemabundu, mamwe ma metabolites asina kurongeka zvakanaka muTIL akapfumawo zvakasiyana (Mifananidzo S3 neS4). Nokudaro, data iri rinoratidza seti inovimbisa ye immunomodulatory metabolites yekutsvagurudza kwakawanda.
(A) Kuwanda kweMNA mumasero eCD4+, CD8+ uye CD45- kubva kumasero e ascites nebundu. Bhokisi rinoratidza huwandu hwepakati (mutsara), huwandu hwepakati pemakota (frame hinge) uye huwandu hwedata, kusvika ka1.5 pane huwandu hwepakati pemakota (frame whisker). Sezvakatsanangurwa muPatient Materials and Methods, shandisa kukosha kwe limma yemurwere kuti uone kukosha kweP (*P<0.05 uye **P<0.01). (B) Dhiyagiramu yeSchematic ye metabolism yeMNA (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. MaEnzayimu (girini): 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 ye scRNA-seq ye ascites (grey) uye bundu (tsvuku; n = varwere 3). (D) Kuonekwa kweNNMT mumasero akasiyana-siyana akaonekwa uchishandisa scRNA-seq. (E) Kuonekwa kweNNMT neAOX1 muSK-OV-3, human embryonic kidney (HEK) 293T, T cells uye MNA-treated T cells. Kuonekwa kwakapetwa kunoratidzwa zvichienderana neSK-OV-3. Patani yekutaura neSEM inoratidzwa (n = 6 vapendi vane hutano). Ct values ​​​​inopfuura 35 inoonekwa seisingaonekwe (UD). (F) Kuonekwa kweSLC22A1 neSLC22A2 muSK-OV-3, HEK293T, T cells neT cells dzakarapwa ne8mM MNA. Kuonekwa kwakapetwa kunoratidzwa zvichienderana neSK-OV-3. Patani yekutaura neSEM inoratidzwa (n = 6 vapeyi vane hutano). Ct values ​​​​inopfuura 35 inoonekwa seisingaonekwe (UD). (G) Zviri muSero MNA muT cells dzine hutano dzakashanda mushure memaawa makumi manomwe nemaviri ekubatanidzwa neMNA. Patani yekutaura neSEM inoratidzwa (n = 4 vapeyi vane hutano).
MNA inogadzirwa nekutamisa boka remethyl kubva kuS-adenosyl-1-methionine (SAM) kuenda kunicotinamide (NA) kuburikidza nenicotinamide N-methyltransferase (NNMT; Mufananidzo 3B). NNMT inoratidzwa zvakanyanya mumhando dzakasiyana dzekenza dzevanhu uye inobatanidzwa nekuwanda, kupinda uye metastasis (25-27). Kuti tinzwisise zviri nani kwakabva MNA mumasero eT muTME, takashandisa scRNA-seq kuratidza kuratidzwa kweNNMT mumarudzi esero muascites nemabundu evarwere vatatu veHGSC (Tafura S5). Kuongororwa kwemasero angangoita 6,500 kwakaratidza kuti munzvimbo dzine ascites nemabundu, kuratidzwa kweNNMT kwakaganhurirwa kune vanhu vanofungidzirwa kuti vane fibroblast nemabundu (Mufananidzo 3, C naD). Zvakakosha kuziva kuti hapana kuratidzwa kweNNMT kuri pachena mune chero huwandu hwevanhu hunoratidza PTPRC (CD45 +) (Mufananidzo 3D naMufananidzo S5A), izvo zvinoratidza kuti MNA yakawanikwa mu metabolite spectrum yakaunzwa mumasero eT. Kuonekwa kwe aldehyde oxidase 1 (AOX1) kunoshandura MNA kuita 1-methyl-2-pyridone-5-carboxamide (2-PYR) kana 1-methyl-4-pyridone-5-carboxamide (4-PYR); Mufananidzo 3B) unongogumirawo kuhuwandu hwe fibroblasts dzinoburitsa COL1A1 (Mufananidzo S5A), izvo pamwe chete zvinoratidza kuti masero eT haana kugona kweMNA metabolism yakajairika. Maitiro ekutaura kwemajini aya ane chekuita neMNA akaongororwa uchishandisa seti yechipiri yedata remasero akazvimiririra kubva kuascites kubva kuvarwere veHGSC (Mufananidzo S5B; n = 6) (16). Pamusoro pezvo, quantitative polymerase chain reaction (qPCR) ongororo yemasero eT ane hutano akarapwa neMNA yakaratidza kuti zvichienzaniswa nemasero eSK-OV-3 ovarian tumor cells, NNMT kana AOX1 zvainge zvisina kuratidzwa (Mufananidzo 3E). Mhedzisiro iyi isingatarisirwi inoratidza kuti MNA inogona kuburitswa kubva kufibroblasts kana tumors kuenda kumasero eT ari pedyo muTME.
Kunyangwe hazvo vanhu vakabvunzwa vanosanganisira mhuri yevanotakurwa nemakemikari kubva pa1 kusvika pa3 (OCT1, OCT2 naOCT3) vakanyorerwa nemhuri yevanotakurwa nemasero 22 (SLC22) (SLC22A1, SLC22A2 naSLC22A3), vanhu vanogona kutakurwa nemasero eMNA havasati vatsanangurwa (28). QPCR yemRNA kubva kumasero eT ane hutano yakaratidza mazinga eSLC22A1 akaderera asi mazinga eSLC22A2 asingaonekwe, izvo zvakasimbisa kuti yakambotaurwa mumabhuku (Mufananidzo 3F) (29). Kusiyana neizvi, SK-OV-3 ovarian tumor cell line yakaratidza mazinga epamusoro emasero ese ari maviri (Mufananidzo 3F).
Kuti zviongororwe kuti masero eT anogona kupinza MNA yekunze, masero eT anopa hutano akarimwa kwemaawa makumi manomwe nemaviri paine huwandu hwakasiyana hweMNA. Pasina MNA yekunze, huwandu hweMNA mumasero hahugone kuonekwa (Mufananidzo 3G). Zvisinei, masero eT akagadziriswa akarapwa neMNA yekunze akaratidza kuwedzera kwehuwandu hweMNA mumasero zvichienderana nedosi, kusvika ku6 mM MNA (Mufananidzo 3G). Mhedzisiro iyi inoratidza kuti pasinei nehuwandu hwakaderera hwe transporter expression uye kushaikwa kwe main enzyme inokonzera intracellular MNA metabolism, TIL inogona kutora MNA.
Kuwanda kwemametabolites mumasero eT evarwere uye kuyedzwa kweMNA in vitro kunowedzera mukana wekuti fibroblasts (CAF) dzinobatanidza cancer dzinoburitsa MNA uye masero ebundu anogona kudzora phenotype uye mashandiro eTIL. Kuti vaone mhedzisiro yeMNA pamasero eT, masero eT anopa hutano akashanda mu vitro kana MNA iripo kana kuti isipo, uye kuwanda kwawo uye kugadzirwa kwecytokine zvakaongororwa. Mushure memazuva manomwe ekuwedzera MNA padosi yepamusoro, huwandu hwevanhu hwakapetwa kaviri hwakaderedzwa zvine mwero, nepo simba rakachengetedzwa padosi dzese (Mufananidzo 4A). Pamusoro pezvo, kurapwa kweMNA yekunze kwakaita kuti huwandu hwemasero eCD4 + neCD8 + T anoratidza tumor necrosis factor-α (TNFα; Mufananidzo 4B). Kusiyana neizvi, kugadzirwa kweIFN-γ mukati memasero kwakaderedzwa zvakanyanya mumasero eCD4 + T, asi kwete mumasero eCD8 + T, uye pakanga pasina shanduko huru mu interleukin 2 (IL-2; Mufananidzo 4, C naD). Saka, enzyme-linked immunosorbent assay (ELISA) yema supernatants kubva muMNA-treated T cell cultures yakaratidza kuwedzera kukuru muTNFα, kudzikira kweIFN-γ, uye kusachinja muIL-2 (Mufananidzo 4, E kusvika G). . Kudzikira kweIFN-γ kunoratidza kuti MNA inogona kuita basa mukudzivisa anti-tumor activity yeT cells. Kuti titevedzere mhedzisiro yeMNA paT cell-mediated cytotoxicity, chimeric antigen receptor T (FRα-CAR-T) cells targeting folate receptor α uye CAR-T (GFP) inodzorwa negreen fluorescent protein (GFP) -CAR-T cells anogadzirwa nehealthy donor peripheral blood mononuclear cells (PBMC). CAR-T cells dzakarimwa kwemaawa makumi maviri nemana pamberi peMNA, uye dzakazorimwa pamwe chete ne human SK-OV-3 ovarian tumor cells dzichiratidza folate receptor α pa effector to target ratio ye10:1. Kurapwa kweMNA kwakaita kuti basa rekuuraya masero eFRα-CAR-T ridzikire zvakanyanya, iro rakafanana nemasero eFRα-CAR-T akarapwa neadenosine (Mufananidzo 4H).
(A) Kuwanda kwemasero anobatsira uye kuwanda kwevanhu (PD) zvakananga kubva mukumera pazuva rechi7. Girafu yebhara inomiririra avhareji + SEM yevapi vemazano vatanhatu vane hutano. Inomiririra data kubva kune angangoita n = 3 bvunzo dzakazvimirira. (B kusvika D) CD3/CD28 neIL-2 zvakashandiswa kumutsa masero eT pahuwandu hwavo hweMNA kwemazuva manomwe. Pamberi pekuongorora, masero akakurudzirwa nePMA/ionomycin neGolgiStop kwemaawa mana. Kuratidzwa kweTNFα (B) mumasero eT. Mufananidzo wemuenzaniso (kuruboshwe) uye data retabhura (kurudyi) reTNFα expression mumasero mapenyu. Kuratidzwa kweIFN-γ (C) uye IL-2 (D) mumasero eT. Kuratidzwa kwemacytokine kwakayerwa ne flow cytometry. Girafu yebhara inomiririra avhareji (n = 6 vapi vemazano vane hutano) + SEM. Shandisa ongororo ye one-way ye variance uye zviyero zvinodzokororwa (*P<0.05 uye **P<0.01) kuti uone kukosha kweP. Inomiririra data kubva kune angangoita n = 3 bvunzo dzakazvimirira. (E kusvika kuG) CD3/CD28 neIL-2 zvakashandiswa kumutsa masero eT pahuwandu hwawo hweMNA kwemazuva manomwe. Nzvimbo iyi yakaunganidzwa maawa mana asati asvika uye mushure mePMA/ionomycin stimulation. Huwandu hweTNFα (E), IFN-γ (F) neIL-2 (G) hwakayerwa neELISA. Bargraph inomiririra avhareji (n = 5 vapenyu vanopa) + SEM. P value yakayerwa uchishandisa one-way analysis ye variance uye yakadzokororwa kuyerwa (*P<0.05). Mutsetse une dotted unoratidza muganho wekuona kwekuona. (H) Cell lysis assay. FRα-CAR-T kana GFP-CAR-T cells dzakagadziriswa ne adenosine (250μM) kana MNA (10 mM) kwemaawa makumi maviri nemana, kana kuti dzakasiyiwa dzisina kurapwa (Ctrl). Kuuraya kwechikamu chemasero eSK-OV-3 kwakayerwa. P value yakayerwa neWelch t test (*P<0.5 uye **P<0.01).
Kuti tinzwisise mashandiro eTNFα expression regulation zvinoenderana neMNA, shanduko muTNFα mRNA yeT-treated T cells dzakarapwa neMNA dzakaongororwa (Mufananidzo 5A). T cells dzine hutano dzakarapwa neMNA dzakaratidza kuwedzera kaviri kweTNFα transcription levels, zvichiratidza kuti MNA inoenderana neTNFα transcriptional regulation. Kuti tiongorore nzira iyi yekudzora, zvinhu zviviri zvinozivikanwa zvetranscription zvinodzora TNFα, zvinoti activated T cell nuclear factor (NFAT) uye specific protein 1 (Sp1), zvakaongororwa zvichienderana neMNA inobatanidza kune proximal TNFα promoter (30). TNFα promoter ine nzvimbo 6 dzakaonekwa dzeNFAT binding uye nzvimbo mbiri dzeSp1 binding, dzinopindirana panzvimbo imwe chete [-55 base pairs (bp) kubva pa5'cap] (30). Chromatin immunoprecipitation (ChIP) yakaratidza kuti painorapwa neMNA, kusunga Sp1 kune TNFα promoter kwakawedzera katatu. Kubatanidzwa kweNFAT kwakawedzerawo uye kwakaswedera pedyo nekukosha (Mufananidzo 5B). Ruzivo urwu runoratidza kuti MNA inotonga kutaurwa kweTNFα kuburikidza neSp1 transcription, uye zvishoma kutaurwa kweNFAT.
(A) Zvichienzaniswa nemasero eT akakurira asina MNA, kuchinja kwekuonekwa kweTNFα mumasero eT akarapwa neMNA. Maitiro ekutaura neSEM anoratidzwa (n = 5 vapi vane hutano). Inomiririra data kubva kuongororo dze n = 3 dzakazvimiririra. (B) Chinosimudzira TNFα chemasero eT akarapwa ne kana asina 8 mM MNA mushure meNFAT neSp1 zvakabatanidzwa ne (Ctrl) uye PMA/ionomycin stimulation kwemaawa mana. Immunoglobulin G (IgG) neH3 zvakashandiswa sezvinodzora zvisina kunaka uye zvakanaka zve immunoprecipitation, zvichiteerana. Kuyera kweChIP kwakaratidza kuti kusunga kweSp1 neNFAT kune chinokurudzira TNFα mumasero akarapwa neMNA kwakawedzera kakawanda zvichienzaniswa nechinodzora. Inomiririra data kubva kuongororo dze n = 3 dzakazvimiririra. P value yakatarwa ne multiple t-tests (*** P <0.01). (C) Zvichienzaniswa ne ascites yeHGSC, masero eT (asiri cytotoxic) akaratidza kuwedzera kwekutaura kweTNF mubundu. Mavara acho anomiririra varwere vakasiyana. Masero akaratidzwa akatorwa zvisina tsarukano kusvika pa300 uye akavhunduka kuti aderedze kudhonza kwakanyanya (** Padj = 0.0076). (D) Muenzaniso weMNA wakapihwa wekenza yemuzai. MNA inogadzirwa mumasero egomarara uye fibroblasts muTME uye inotorwa nemasero eT. MNA inowedzera kubatana kweSp1 kune TNFα promoter, zvichikonzera kuwedzera kweTNFα transcription uye TNFα cytokine production. MNA inokonzerawo kudzikira kweIFN-γ. Kudziviswa kwebasa reT cell kunotungamira mukudzikira kwekugona kuuraya uye kukura kwegomarara nekukurumidza.
Sekureva kwemishumo, TNFα ine mhedzisiro yekurwisa bundu nekumashure inoenderana nekumberi nekumashure, asi ine basa rinozivikanwa mukusimudzira kukura nekupararira kwekenza yemuzai (31-33). Sekureva kwemishumo, kuwanda kweTNFα muascites nematishu ebundu muvarwere vane kenza yemuzai kwakakwira kupfuura kuri mumatishu asina njodzi (34-36). Panyaya yekushanda kwayo, TNFα inogona kudzora kushanda, kushanda uye kuwanda kwemasero machena eropa, uye kuchinja phenotype yemasero ekenza (37, 38). Zvichienderana nezvakawanikwa izvi, ongororo ye differential gene expression yakaratidza kuti TNF yakawedzera zvakanyanya mumatishu eT mumatishu ebundu zvichienzaniswa neascites (Mufananidzo 5C). Kuwedzera kwekutaura kweTNF kwaingoonekwa chete mumatishu eT ane phenotype isina cytotoxic (Mufananidzo S5A). Mukupfupisa, data iri rinotsigira maonero ekuti MNA ine mhedzisiro mbiri dzekudzivirira immune uye dzinokurudzira bundu muHGSC.
Kuisa mavara echiedza chefluorescent zvichibva pa flow cytometry kwave nzira huru yekudzidza metabolism yeTIL. Zvidzidzo izvi zvakaratidza kuti zvichienzaniswa ne peripheral blood lymphocytes kana T cells kubva ku secondary lymphoid organs, murine ne human TIL vane katsika kekutora glucose (4, 39) uye kurasikirwa zvishoma nezvishoma kwebasa re mitochondrial (19, 40). Kunyangwe takaona mhedzisiro yakafanana muchidzidzo ichi, budiriro huru ndeyekuenzanisa metabolism yemasero e tumor neTIL kubva ku tumor tissue imwechete yakabviswa. Zvichienderana nemimwe yemishumo yapfuura, tumor (CD45-EpCAM +) masero kubva ku ascites nema tumor ane glucose yakakwira kupfuura CD8 + neCD4 + T cells, zvichitsigira kuti glucose yakakwira yemasero e tumor inogona kuenzaniswa neT cells. Pfungwa yemakwikwi eT cell. TME. Zvisinei, mitochondrial activity yemasero e tumor yakakwira kupfuura yeCD8 + T cells, asi mitochondrial activity yakafanana neyeCD4 + T cells. Izvi zvinosimbisa dingindira riri kubuda rekuti oxidative metabolism yakakosha kumasero e tumor (41, 42). Vanotaurawo kuti masero eCD8 + T anogona kunge ari nyore kubatwa ne oxidative dysfunction kupfuura masero eCD4 + T, kana kuti masero eCD4 + T anogona kushandisa carbon sources kunze kweglucose kuchengetedza mitochondrial activity (43, 44). Zvinofanira kucherechedzwa kuti hatina kuona musiyano mukutorwa kweglucose kana mitochondrial activity pakati peCD4 + T effectors, T effector memory uye T central memory cells mu ascites. Saizvozvowo, differentiation state yeCD8 + T cells mu tumors haina chekuita nekuchinja mukutorwa kweglucose, zvichiratidza musiyano mukuru pakati pemasero eT akakurira mu vitro ne human TIL in vivo (22). Izvi zvakaonekwawo zvakasimbiswa nekushandiswa kwe unbiased automatic cell population allocation, izvo zvakaratidza zvakare kuti masero eCD45 + / CD3- / CD4 + / CD45RO + ane glucose uptake yakakwira uye mitochondrial activity kupfuura masero e tumour anowanikwa asi ane Metabolic active cell population. Huwandu uhu hunogona kumiririra huwandu hwemasero e myeloid suppressor kana plasmacytoid dendritic activity akaonekwa mu scRNA-seq analysis. Kunyangwe zvese izvi zvakambotaurwa nezvemabundu emumazai evanhu [45], zvichiri kuda basa rakawanda nderekutsanangura huwandu hwemasero aya eropa.
Kunyange zvazvo nzira dzinoshandiswa pakuita flow cytometry dzichigona kujekesa mutsauko mukuru mu glucose uye oxidative metabolism pakati pemhando dzemasero, metabolites chaiyo inogadzirwa neglucose kana zvimwe zvinhu zvekabhoni zve metabolism ye mitochondrial mu TME haisati yawanikwa. Kupa kuvapo kana kusavapo kwe metabolites kune imwe subset yeTIL kunoda kucheneswa kwehuwandu hwemasero kubva munyama yakabviswa. Nokudaro, nzira yedu yekuvandudza masero pamwe chete ne mass spectrometry inogona kupa ruzivo nezve metabolites dzakapfuma zvakasiyana mumasero eT uye mapoka emasero egomarara mukuenzanisa sampuli dzevarwere. Kunyangwe nzira iyi iine zvakanakira kupfuura fluorescence-activated cell sorting, mamwe maraibhurari e metabolites anogona kukanganiswa nekuda kwekugadzikana kwemukati uye/kana kukurumidza turnover rate (22). Kunyange zvakadaro, nzira yedu yakakwanisa kuona metabolites mbiri dzinozivikanwa dze immunosuppressive, adenosine ne kynurenine, nekuti dzakasiyana zvikuru pakati pemhando dzesamples.
Kuongorora kwedu metabonomic yemabundu uye TIL subtypes kunopa ruzivo rwakawanda nezvebasa re metabolites mu ovarian TME. Chekutanga, tichishandisa flow cytometry, takaona kuti pakanga pasina musiyano mu mitochondrial activity pakati pebundu neCD4 + T cells. Zvisinei, LC-MS/MS analysis yakaratidza shanduko huru mukuwanda kwema metabolites pakati pevanhu ava, zvichiratidza kuti mhedziso nezve TIL metabolism uye basa rayo rese re metabolic rinoda kududzirwa kwakanyatsojeka. Chechipiri, MNA ndiyo metabolite ine musiyano mukuru pakati peCD45-cells neT cells mu ascites, kwete bundu. Nokudaro, compartmentalization uye bundu nzvimbo zvinogona kuva nemigumisiro yakasiyana pa TIL metabolism, izvo zvinoratidza kusiyana kunogona kuitika mune microenvironment yakapihwa. Chechitatu, kutaura kweMNA-producing enzyme NNMT kunonyanya kuganhurirwa kuCAF, inova bundu masero zvishoma, asi MNA levels dzinoonekwa dzinoonekwa mu T cells dzinobva bundu. Kuwanda kweNNMT mu ovarian CAF kune mhedzisiro inozivikanwa yekukurudzira kenza, chimwe chikamu nekuda kwekusimudzirwa kweCAF metabolism, kupinda bundu uye metastasis (27). Kunyangwe huwandu hwese hweTIL huri pakati nepakati, kuratidzwa kweNNMT muCAF kwakabatana zvakanyanya neCancer Genome Atlas (TCGA) mesenchymal subtype, iyo inobatanidzwa nekutadza kufambira mberi kwechirwere (27, 46, 47). Chekupedzisira, kuratidzwa kweenzyme AOX1 inokonzera kuora kweMNA kwakaganhurirwawo kuvanhu veCAF, izvo zvinoratidza kuti masero eT haana kugona kugaya MNA. Mhedzisiro iyi inotsigira pfungwa yekuti kunyangwe paine basa rakawanda rinodiwa kuti zviongororwe kuwanikwa uku, huwandu hwakawanda hweMNA mumasero eT hunogona kuratidza kuvapo kwenzvimbo diki yeCAF inodzvinyirira immune system.
Zvichienderana nehuwandu hushoma hwezvinhu zvinotakura MNA uye huwandu husingaonekwe hwemapuroteni akakosha anobatanidzwa mu metabolism yeMNA, kuvapo kweMNA mumasero eT hazvitarisirwi. NNMT kana AOX1 hazvina kuonekwa nekuongorora kwe scRNA-seq uye qPCR yakatarwa yemapoka maviri akazvimiririra. Mhedzisiro iyi inoratidza kuti MNA haina kugadzirwa nemasero eT, asi inotorwa kubva kuTME yakapoteredza. Kuedza kwe in vitro kunoratidza kuti masero eT anowanzo unganidza MNA yekunze.
Zvidzidzo zvedu zve in vitro zvakaratidza kuti exogenous MNA inokonzera kuratidzwa kweTNFα mumasero eT uye inowedzera kubatana kweSp1 kune TNFα promoter. Kunyangwe TNFα iine mabasa ekurwisa bundu uye ekurwisa bundu, mukenza ye ovarian, TNFα inogona kukurudzira kukura kwekenza ye ovarian (31-33). Kuderedza TNFα mukukura kwemasero e ovarian tumor kana kubviswa kwechiratidzo cheTNFα mumhando dzembeva kunogona kuvandudza kugadzirwa kwe cytokine inorwisa TNFα uye kudzivirira kukura kwebundu (32, 35). Nokudaro, muchiitiko ichi, TME-derived MNA inogona kushanda se pro-inflammatory metabolite kuburikidza neTNFα-dependent mechanism kuburikidza ne autocrine loop, nokudaro ichikurudzira kuitika nekupararira kwekenza ye ovarian (31). Zvichibva pamukana uyu, TNFα blockade iri kudzidzwa semushonga unogona kurapa kenza ye ovarian (37, 48, 49). Pamusoro pezvo, MNA inokanganisa cytotoxicity yemasero eCAR-T kumasero e ovarian tumor, zvichipa humwe humbowo hwekudzvinyirirwa kweMNA-mediated immune. Pamwe chete, mhedzisiro iyi inoratidza muenzaniso umo bundu nemasero eCAF zvinoburitsa MNA mu extracellular TME. Kuburikidza ne (i) TNF-induced ovarian cancer stimulation uye (ii) MNA-induced T cell cytotoxic activity inhibition, izvi zvinogona kuva nemhedzisiro yebundu mbiri (Mufananidzo 5D).
Mukupedzisa, nekushandisa musanganiswa wekukurumidza kuwedzera masero, kutevedzana kwemasero ega uye kuongorora metabolic, chidzidzo ichi chakaratidza mutsauko mukuru we immunometabolomic pakati pemabundu nemasero e ascites muvarwere veHGSC. Ongororo iyi yakazara yakaratidza kuti kune mutsauko mukutorwa kweglucose uye mitochondrial activity pakati pemasero eT, uye yakaratidza MNA semetabolite inotonga immune system isiri yemasero. Data iri rine chekuita nekuti TME inokanganisa sei metabolism yemasero eT mukenza yevanhu. Kunyangwe kukwikwidzana kwakananga kwezvinovaka muviri pakati pemasero eT nemasero ekenza kwakataurwa, metabolites inogonawo kushanda sevatongi vasina kunanga kukurudzira kufambira mberi kwebundu uye pamwe kudzvinyirira mhinduro dzemuviri dzekudzivirira. Tsananguro yakawedzerwa yebasa remametabolites aya anogona kuvhura dzimwe nzira dzekusimudzira mhinduro yekudzivirira immune system yekurwisa bundu.
Zviyero zvevarwere neruzivo rwekiriniki zvakawanikwa kuburikidza neBC cancer tumor tissue repository repository yakasimbiswa neCanadian Tissue Repository Network. Zvichienderana neprotocol yakatenderwa neBC Cancer Research Ethics Committee neYunivhesiti yeBritish Columbia (H07-00463), zviyero zvese zvevarwere neruzivo rwekiriniki zvakawana mvumo yakanyorwa zvine ruzivo kana kuti zvakarega mvumo yavo zviri pamutemo. Zviyero izvi zvinochengetwa muBioBank (BRC-00290) yakatenderwa. Hunhu hwakadzama hwemurwere hunoratidzwa muTables S1 neS5. Pakuchengetedza masero, scalpel inoshandiswa kuomesa sampuli yegomarara remurwere uye yobva yasundidzirwa nepafirita ye100-micron kuti iwane kumiswa kwesero rimwe chete. Ascites yemurwere yakatenderedzwa ne1500 rpm kwemaminitsi gumi pa4°C kuti iparadze masero uye ibvise supernatant. Masero akawanikwa kubva mubundu neascites akachengetwa mu50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) uye 10% dimethyl sulfoxide. Ma suspensions aya akachengetedzwa e single cell akanyungudutswa uye akashandiswa pakutsvaga metabolomics uye metabolite yakatsanangurwa pazasi.
Mushonga wacho une 0.22 μm yakaseferwa 50:50 yakawedzerwa RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) yakawedzerwa ne 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) uye 50 μMB-mercaptoethanol. AimV (Invitrogen) yakawedzerwa ne 20 mM Hepes (Thermo Fisher Scientific) uye 2 mM l-glutamine (Thermo Fisher Scientific). Flow cytometer staining buffer yaive ne 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) yakawedzerwa ne 3% heat-inactivated AB human serum (Sigma). Iyo masero anovandudza buffer inoumbwa ne 0.22μm filtered PBS uye inowedzerwa ne 0.5% heat-inactivated human AB serum (Sigma-Aldrich).
Mukati mekupisa kwe37°C, masero akaiswa 10 nM MT DR uye 100 μM 2-NBDG kwemaminitsi makumi matatu. Tevere, masero akaiswa dye yekurarama eF506 pa4°C kwemaminitsi gumi nemashanu. Dzorerai masero muFC Block (eBioscience) neBrilliant Stain Buffer (BD Biosciences), sanganisai flow cytometer staining buffer (zvichienderana nemirairo yemugadziri), uye garai muchitonhorera kwemaminetsi gumi patembiricha yemukamuri. Dzorerai masero neseti yema antibodies (Table S2) mu flow cytometry staining buffer pa4°C kwemaminitsi makumi maviri. Dzorerai masero mu flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) musati maongorora. Shandisai SpectroFlo neFlowJo V10 kuongorora data rekuverenga masero, uye shandisai GraphPad Prism 8 kugadzira data. Kuwanda kwechiedza chepakati (MFI) che2-NBDG neMT DR kwakagadziridzwa kuti kuve kwakajairika, uye bvunzo yeT yakazoshandiswa pakuongorora nhamba kuti iverenge varwere vakaenzana. Bvisa vanhu vese vane zviitiko zvisingasviki makumi mana kubva mukuongorora; isa MFI value ye1 kune chero negative values ​​​​usati waita ongororo yenhamba uye kuona data.
Kuti tiwedzere nzira yekubata magating yepaneru iri pamusoro apa, takashandisa tsananguro yakazara yemuti wekugadzirisa chimiro (FAUST) (21) kuti tipe masero otomatiki kuvanhu mushure mekubvisa masero akafa muFlowJo. Isu tinotarisira kubuda kwemasero nemaoko kuti tisanganise vanhu vanoita sevakasarongerwa (tichibatanidza PD1+ nemasero ePD1-tumor) uye vanhu vakachengetwa. Sampuro yega yega ine avhareji yemasero anopfuura 2%, kune vanhu gumi nerimwe chete.
Kushandiswa kweFicoll gradient density centrifugation kuparadzanisa PBMC kubva ku leukocyte separation products (STEMCELL Technologies). Masero eCD8 + T akaparadzaniswa kubva kuPBMC achishandisa CD8 MicroBeads (Miltenyi) uye akawedzerwa mu complete medium achishandisa TransAct (Miltenyi) kwemavhiki maviri zvichienderana nemirairo yemugadziri. Masero akabvumirwa kumira kwemazuva mashanu mu complete medium ine IL-7 (10 ng/ml; PeproTech), uye akazomutswazve neTransAct. Pazuva rechinomwe, zvichienderana nemirairo yemugadziri, human CD45 MicroBeads (Miltenyi) yakashandiswa kupfumisa masero mumarounds matatu akatevedzana. Masero akaverengerwa kuti aongorore flow cytometry (sezvatsanangurwa pamusoro), uye miriyoni imwe yemasero akaverengerwa katatu kuti aongorore LC-MS/MS. Samples dzakagadziriswa neLC-MS/MS sezvakatsanangurwa pazasi. Takafungidzira huwandu hwemetabolite husipo nenhamba yeion ye1,000. Sampuro yega yega inogadziriswa nenhamba yehuwandu hwemaion (TIC), inoshandurwa ne logarithmically uye inogadziriswa otomatiki muMetaboAnalystR isati yaongororwa.
Sero rimwe chete remurwere wega wega rakanyungudutswa ndokusefa kuburikidza nefirita ye40 μm kuita medium yakazara (sezvatsanangurwa pamusoro). Zvichienderana neprotocol yemugadziri, madenderedzwa matatu akatevedzana ekusarudza kwakanaka kuburikidza nekupatsanurwa kwemabhedhi emagnetic uchishandisa MicroBeads (Miltenyi) akashandiswa kuwedzera sampuli dzeCD8+, CD4+ uye CD45- cells (pachando). Muchidimbu, masero anodzoserwa mu cell enrichment buffer (sezvatsanangurwa pamusoro) uye akaverengwa. Masero akaiswa maCD8 beads evanhu, maCD4 beads evanhu kana maCD45 beads evanhu (Miltenyi) pa4°C kwemaminitsi gumi nemashanu, uye ozogezwa ne cell enrichment buffer. Sampuro inopfuudzwa kuburikidza neLS column (Miltenyi), uye positive nenegative fractions zvinounganidzwa. Kuti kuderedze nguva uye kuwedzera danho rekupora kwemasero, CD8-fraction inobva yashandiswa kudenderedzwa rechipiri reCD4+ enrichment, uye CD4-fraction inoshandiswa kuCD45-enrichment inotevera. Chengeta mhinduro pachando mukati menguva yese yekuparadzanisa.
Kugadzirira masampuli ekuongororwa kwemametabolite, masero akagezwa kamwe chete nemusanganiswa wemunyu unotonhora, uye 1 ml ye80% methanol yakawedzerwa kune imwe neimwe sample, yozobviswa vortex uye yoiswa mu liquid nitrogen. Masampuli akagadziriswa katatu nechando-nyunguduka uye akaiswa centrifuge pa14,000 rpm kwemaminitsi gumi nemashanu pa4°C. Supernatant ine metabolite inopwanyika kusvika yaoma. Mametabolite akanyungudutswa zvakare mu50 μl ye0.03% formic acid, obva asanganiswa, uye obva aiswa centrifuge kubvisa marara.
Bvisa metabolites sezvakatsanangurwa pamusoro apa. Chinja supernatant kubhodhoro remvura rine chromatography rinoita basa rakakura kuti utsvage metabolomics. Shandisa nzira yekurapa isina kurongeka kuti ubatise sample yega yega nenhamba yakafanana yemasero kudzivirira migumisiro yebatch. Takaita ongororo yequalitative ye global metabolites yakamboburitswa paAB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Kuongororwa kweChromatographic uye kubatanidzwa kwenzvimbo yepamusoro kwakaitwa uchishandisa MultiQuant version 2.1 software (Applied Biosystems SCIEX).
Nhamba yemaion ye1000 yakashandiswa kufungidzira kukosha kwemetabolite isipo, uye TIC yemuenzaniso wega wega yakashandiswa kuverenga nzvimbo yakajairwa yepamusoro yemetabolite yega yega yakawanikwa kugadzirisa shanduko dzakaunzwa nekuongorora kwezviridzwa kubva mukugadzirisa sampuro. Mushure mekunge TIC yajairwa, MetaboAnalystR(51) (default parameter) inoshandiswa pakushandura logarithmic uye otomatiki norm line scaling. Takashandisa PCA ne vegan R package kuita ongororo yekuongorora misiyano yemetabolome pakati pemhando dzesamples, uye takashandisa partial redundancy analysis kuongorora varwere. Shandisa nzira yeWard kugadzira heat map dendrogram kuti tiunganidze daro reEuclidean pakati pemasamples. Takashandisa limma (52) pahuwandu hwemetabolite hwakajairwa kuti tione metabolites dzakawanda zvakasiyana mumhando yese yemasero uye microenvironment. Kuti zvive nyore kutsanangura, tinoshandisa group mean parameter kuti titsanangure modhi, uye tifunge nezvemhando dzemasero mu microenvironment seboka rega rega (n = 6 groups); Pakuyedzwa kwekukosha, takaita zviyero zvitatu zvakadzokororwa zvemetabolite yega yega. Kuti tidzivise kudzokororwa kwenhema, murwere akabatanidzwa sechipingamupinyi mukugadzirwa kwelimma. Kuti titarise mutsauko uripo mumetabolite pakati pevarwere vakasiyana, takagadzirisa modhi yelimma kusanganisira varwere nenzira yakatarwa. Tinoshuma kukosha kwemusiyano wakafanotaurwa pakati pemhando yesero nenzvimbo diki yePadj <0.05 (Benjamini-Hochberg correction).
Mushure mekuwedzerwa simba remuviri tichishandisa Miltenyi Dead Cell Removal Kit (>80% viability), kutevedzana kwetranscriptome yemasero mamwechete kwakaitwa pamasero ese akarembera akaomeswa nechando uye sampuli dzematumbu uchishandisa protocol ye10x 5′gene expression. Nyaya shanu dzine tumors neascites dzakafanana dzakaongororwa, kunyangwe kusava nesimba kwemuviri kubva pasampuli imwe chete yetumo kwakadzivirira kuiswa kwayo. Kuti tiwane sarudzo dzakawanda dzevarwere, takabatanidza sampuli dzemurwere wega wega munzira dze10x chromium controller, uye takaongorora nzvimbo dzematumbu zvakasiyana. Mushure mekutevera [Illumina HiSeq 4000 28×98 bp paired end (PE), genome yeQuebec; avhareji yekuverenga 73,488 uye 41,378 pasero rega rega regomarara neascites zvichiteerana], takashandisa CellSNP naVireo (53) (zvichibva paCellSNP sezvo SNP yemunhu yakajairika (VCF) inopihwa neGRCh38 inopihwa humbowo hwemupi. Tinoshandisa SNPRelate kuti tifunge humbowo hwepedyo (IBS) hwemamiriro e genotype yemurwere (IBS), tichibvisa masero asina kugoverwa nemasero akaonekwa semaduplexes uye tichifananidza vanopa pakati peascites nemasampuli egomarara (54). Tichishandisa basa iri, takachengeta zviitiko zvitatu zvine huwandu hwakawanda hwemasero mugomarara neascites kuti tiongorore pasi. Mushure mekuita danho rekusefa mass muscater (55) uye scran (56) BioConductor packaging, izvi zvakaburitsa masero 6975 (masero 2792 ne4183 kubva kugomarara neascites, zvichiteerana) kuti tiongorore. Tinoshandisa igraph's (57) Louvain clustering ye shared nearest neighbor network (SNN) zvichibva paJaccard. daro kubva kumasero eboka zvichienda kune mamwe ma cluster cells. Ma cluster acho akanyorwa nemaoko kumarudzi emasero anofungidzirwa zvichibva pakugadzirwa kwemajini emarker uye akaonekwa ne t-SNE. Masero eT ane chepfu anotsanangurwa nekugadzirwa kweCD8A neGZMA, kusabatanidza ma subclusters ane ribosomal protein expression yakaderera. Takawana data rakabudiswa naIzar et al. (16), kusanganisira t-SNE embedding yavo, inogona kudzora kupindirana kwekutaura pakati pe immune cell markers nekugadzirwa kweNNMT.
PBMC yakaparadzaniswa kubva ku leukocyte separation products (STEMCELL Technologies) neFicoll gradient density centrifugation. Masero eCD3 + akaparadzaniswa kubva kuPBMC achishandisa ma CD3 beads (Miltenyi). Kana paine kana pasina MNA, masero eCD3+ akashanda ne plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) uye IL-2 (300 U/ml; Proleukin). Pazuva rekupedzisira rekuwedzera, viability (Fixable Viability Dye eFluor450, eBioscience) uye proliferation (123count eBeads, Thermo Fisher Scientific) zvakaongororwa ne flow cytometry. Ongorora mashandiro e effector nekukurudzira masero ane PMA (20 ng/ml) uye ionomycin (1μg/ml) neGolgiStop kwemaawa mana, uye tarisa CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) uye TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Kurudzira qPCR neChIP masero nePMA (20 ng/ml) uye ionomycin (1μg/ml) kwemaawa mana. ELISA supernatant yakaunganidzwa isati yakurudzirwa uye mushure mekukurudzira nePMA (20 ng/ml) uye ionomycin (1 μg/ml) kwemaawa mana.
Tevedzera protocol yemugadziri kuti uparadzanise RNA uchishandisa RNeasy Plus Mini Kit (QIAGEN). Shandisa QIAshredder (QIAGEN) kuti ugadzire sampuro yakafanana. Shandisa RNA ine simba rakawanda (Thermo Fisher Scientific) kugadzira DNA inosanganisa (cDNA). Shandisa TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) kuti uverenge huwandu hwemajini (zvichienderana neprotocol yemugadziri) uchishandisa ma probes anotevera: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] uye Hs01010726_m1 (SLC22A2). Masampuleti aya akashandiswa paStepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) muMicroAmp fast optical 96-well reaction plate (Applied Biosystems) ine MicroAmp optical film. Chero Ct value inodarika 35 inoonekwa seyakakwirira kupfuura chiyero chekuona uye yakanyorwa seisingaonekwe.
Ita ChIP sezvakatsanangurwa kare (58). Muchidimbu, masero akarapwa ne formaldehyde (kuwanda kwekupedzisira 1.42%) uye akaiswa patembiricha yemumba kwemaminitsi gumi. Shandisa buffer yekuwedzera kuzvimba (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl uye 0.1% NP-40) pachando kwemaminitsi gumi, wozoiisa zvakare mu immunoprecipitation buffer sezvakatsanangurwa (58). Sampuro yakazoiswa sonic nematanho anotevera: 10 cycles (20 1-second pulses) uye nguva isingachinji yemasekondi makumi mana. Incubate ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) uye SP1 (Cell Signaling Technology; 3μl) antibodies nemuenzaniso pa4°CC shake husiku hwese. Isa mabheya eprotein A (Thermo Fisher Scientific) nemuenzaniso pa4°C uchizunza zvinyoro nyoro kweawa imwe chete, wobva washandisa chelex beads (Bio-Rad) kuti uwedzere DNA, uye shandisa proteinase K (Thermo Fisher) pakugaya mapuroteni. TNFα promoter yakaonekwa nePCR: forward, GGG TAT CCT TGA TGC TTG TGT; pane zvinopesana, GTG CCA ACA ACT GCC TTT ATA TG (207-bp product). Mifananidzo yakagadzirwa neImage Lab (Bio-Rad) uye yakayerwa uchishandisa software yeImageJ.
Sero repamusoro-soro rema cell rakaunganidzwa sezvakatsanangurwa pamusoro apa. Kuongororwa kwakaitwa zvichienderana nemaitiro emugadziri we human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) uye human IFN-γ ELISA kit (Abcam). Zvichienderana neprotocol yemugadziri, supernatant yakaderedzwa 1:100 kuti ione TNFα neIL-2, uye 1:3 kuti ione IFN-γ. Shandisa EnVision 2104 Multilabel Reader (PerkinElmer) kuyera absorbance pa450 nm.
PBMC yakaparadzaniswa kubva ku leukocyte separation products (STEMCELL Technologies) neFicoll gradient density centrifugation. Masero eCD3 + akaparadzaniswa kubva kuPBMC achishandisa maCD3 beads (Miltenyi). Kana paine kana pasina MNA, masero eCD3+ akashanda ne plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) uye IL-2 (300 U/ml; Proleukin) kwemazuva matatu. Mushure memazuva matatu, masero akaunganidzwa ndokugezwa ne 0.9% saline, uye pellet yakabviswa chando. Kuverengwa kwemasero kwakaitwa ne flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) uchishandisa 123count eBeads.
Zvimedu zvekubvisa zvakasanganiswa sezvakatsanangurwa pamusoro apa. Chimedu chakaomeswa chakagadziriswazve pahuwandu hwe4000 cell equivalents/μl. Ongorora sampuro uchishandisa reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) uye CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), umo electrospray ionization inoshanda mu positive mode. Mobile phase A i 0.1% formic acid (mu H2O), mobile phase B i 90% acetonitrile, 0.1% formic acid. LC gradient inotora 0 kusvika 2 maminitsi e100% A, 2 kusvika 7.1 maminitsi e99% B, uye 7.1 kusvika 8 maminitsi e99% B. Wobva waenzanisa zvakare koramu ne mobile phase A pa flow rate ye 0.6 ml/min kwemaminitsi matatu. . flow rate iri 0.4ml/min, uye column chamber inodziiswa kusvika 50°C. Shandisa MNA's pure chemical standard (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) kuti uise nguva yekuchengetedza (RT) uye kushanduka (RT = 0.882 maminitsi, kushanduka 1 = 137→94.1, kushanduka 2 = 137→92, kushandurwa 3 = 137→78). Kana kushanduka kwese kutatu kwaitika panguva chaiyo yekuchengetedza, transition 1 inoshandiswa pakuenzanisa kuti ive nechokwadi chekuti zvinhu zvacho zvakananga. Mutsetse weMNA (Toronto Research Chemical Company) wakagadzirwa nekusanganiswa kwemasero matanhatu emushonga wemuto (1 mg/ml) kuti uwane zviyero zve 0.1, 1.0, 10 uye 100 ng/ml uye 1.0 uye 10μg/ml zvichiteerana. Muganho wekuona i1 ng/ml, uye mhinduro yemutsara iri pakati pe10 ng/ml ne10μg/ml. Jekiseni rega rega rema microliters maviri emuenzaniso nechiyero rinoshandiswa pakuongorora LC/MS, uye sampuro yekudzora mhando yakasanganiswa inoitiswa majekiseni masere ega ega kuti ive nechokwadi chekugadzikana kwepuratifomu yekuongorora. Mhinduro dzeMNA dzemasample ese emasero akarapwa neMNA dzaive mukati memutsara wemutsara wekuyedza. Kuongorora data kwakaitwa uchishandisa MassHunter quantitative analysis software (v9.0, Agilent).
Chivako chechipiri che αFR-CAR chakagadzirwa kubva kuna Song et al. (59). Muchidimbu, chivakwa chacho chine zvinotevera: CD8a leader sequence, human αFR-specific single-chain variable fragment, CD8a hinge and transmembrane region, CD27 intracellular domain uye CD3z intracellular domain. CAR sequence yakazara yakagadzirwa neGenScript, uye yakazoiswa muchikamu chechipiri che lentiviral expression vector kumusoro kweGFP expression cassette inoshandiswa kuongorora kushanda kwe transduction.
Lentivirus inogadzirwa nekuisa HEK293T cells [American Type Culture Collection (ATCC); inokura muDulbecco's modified Eagle medium ine 10% fetal bovine serum (FBS) uye 1% PenStrep, uye yakashandisa CAR-GFP vector uye The packaging plasmids (psPAX2 na pMD2.G, Addgene) inoshandisa lipofection amine (Sigma-Aldrich). Supernatant ine hutachiona yakatorwa maawa 48 ne72 mushure mekuisirwa, yakaseferwa, uye yakasanganiswa ne ultracentrifugation. Chengeta supernatant yakasanganiswa pa -80°C kusvika yaiswa.
PBMC inoparadzaniswa kubva kuzvigadzirwa zve leukocyte separation zvine hutano (STEMCELL Technologies) neFicoll gradient density centrifugation. Shandisa positive selection CD8 microbeads (Miltenyi) kuti ubvise masero eCD8+ kubva kuPBMC. Kurudzira masero eT neTransAct (Miltenyi) uye muTexMACS medium [Miltenyi; inowedzerwa ne3% heat-inactivated human serum, 1% PenStrep uye IL-2 (300 U/ml)]. Maawa makumi maviri nemana mushure mekukurudzirwa, masero eT akaiswa lentivirus (10 μl concentrated virus supernatant pamasero 106). Mazuva 1 kusvika matatu mushure mekuwedzerwa paCytek Aurora (paFSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), ongorora kuratidzwa kweGFP kwemasero kuratidza kushanda kwe transduction kweinenge 30%.
Masero eCAR-T akarimwa kwemaawa makumi maviri nemana muImmunocult (STEMCELL Technologies; akawedzerwa ne1% PenStrep) pasi pemamiriro anotevera: asina kurapwa, akarapwa ne250 μM adenosine kana 10 mM MNA. Mushure mekurapwa, masero eCAR-T akagezwa nePBS ndokusanganiswa nemasero eSK-OV-3 20,000 [ATCC; muMcCoy 5A medium (Sigma-Aldrich) akawedzerwa ne10% FBS uye 1% PenStrep pa10: Chiyero che effector to target che1 chakawedzerwa mu triplicate muImmunocult medium yakawedzeredzwa. Masero eSK-OV-3 nemasero eSK-OV-3 akasanganiswa ne digitalis saponin (0.5mg/ml; Sigma-Aldrich) akashandiswa senzira dzekudzivirira dzakaipa nedzakanaka, zvichiteerana. Mushure memaawa makumi maviri nemana ekurima pamwe chete, supernatant yakatorwa uye lactate dehydrogenase (LDH) yakayerwa zvichienderana nemirayiridzo yemugadziri (LDH Glo Cytotoxicity Assay Kit, Promega). Supernatant yeLDH yakaderedzwa 1:50 muLDH buffer. Chikamu chekuuraya chakayerwa uchishandisa fomura inotevera: chikamu chekuuraya = chikamu chekugadzirisa / chiyero chikuru chekuuraya x 100%, apo chikamu chekugadzirisa = masero e-co-culture-T chete, uye chiyero chikuru chekuuraya = kudzora-negative.
Sezvinotsanangurwa muchinyorwa kana zvinhu uye nzira, shandisa GraphPad Prism 8, Microsoft Excel kana R v3.6.0 kuti uongorore nhamba. Kana sampuli dzakawanda dzikatorwa kubva kumurwere mumwe chete (senge ascites nebundu), tinoshandisa bvunzo ye paired t kana kusanganisira murwere semhedzisiro isina kurongeka mumuenzaniso we linear kana generalized sezvakakodzera. Pakuongorora metabolomics, bvunzo yekukosha inoitwa katatu.
Kuti uwane zvimwe zvinyorwa zvechinyorwa chino, ndapota tarisai 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. De Bellar Jones (G.Russell. G. Jones), Phineas T. Hamilton (Phineas T.
MNA inobatsira mukudzvinyirirwa kwemasero eT mumuviri uye inomiririra chinangwa chekurapa kenza yevanhu.
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. De Bellar Jones (G.Russell. G. Jones), Phineas T. Hamilton (Phineas T.
MNA inobatsira mukudzvinyirirwa kwemasero eT mumuviri uye inomiririra chinangwa chekurapa kenza yevanhu.
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