Kuongororwa kwakasiyana kwepulmonary nodules inozivikanwa nekomputa tomography (CT) inoramba iri dambudziko mukuita kwekiriniki.Pano, tinoratidzira metabolome yepasi rose ye480 serum samples, kusanganisira kudzora hutano, benign lung nodules, uye stage I lung adenocarcinoma.Adenocarcinomas inoratidza akasiyana metabolomic profiles, nepo benign nodules uye vanhu vane hutano vane hukuru hwakafanana mumaprofiles emetabolomic.Muboka rekutsvaga (n = 306), seti ye 27 metabolites yakaonekwa kuti isiyanise pakati pemanodule asina kunaka uye akaipa.Iyo AUC yemhando yekusarura mukusimbisa kwemukati (n = 104) uye yekusimbisa kwekunze (n = 111) mapoka aive 0.915 uye 0.945, zvichiteerana.Ongororo yenzira yakaratidza yakawedzera glycolytic metabolites yakabatana neyadzikira tryptophan mumapapu adenocarcinoma serum kana ichienzaniswa nebenign nodules uye ine hutano kutonga, uye yakaratidza kuti tryptophan uptake inosimudzira glycolysis mumaseru ekenza yemapapu.Chidzidzo chedu chinosimbisa kukosha kweserum metabolite biomarkers mukuongorora njodzi yemapulmonary nodules akaonekwa neCT.
Kuongororwa kwekutanga kwakakosha kuvandudza huwandu hwekupona kwevarwere vegomarara.Mhedzisiro kubva kuUS National Lung Cancer Screening Trial (NLST) uye European NELSON Chidzidzo chakaratidza kuti kuongorora ne-low-dose computed tomography (LDCT) kunogona kuderedza zvakanyanya kufa kwekenza yemapapu mumapoka ane njodzi yakanyanya1,2,3.Kubva kushandiswa kwakapararira kweLDCT yekuongorora kenza yemapapu, chiitiko chechiitiko chechiitiko chekutsvaga kweasymptomatic pulmonary nodules yakaramba ichiwedzera 4.Pulmonary nodules inotsanangurwa sefocal opacities kusvika 3 cm mudhayamita 5.Isu tinotarisana nekuomerwa mukuongorora mukana wehuipi uye kubata nehuwandu hukuru hwemapulmonary nodules akaonekwa zvakangoerekana zvaitika paLDCT.Kuganhurirwa kweCT kunogona kutungamirira kuongororo dzenguva dzose dzekutevera uye mhedzisiro yenhema, zvichiita kuti kupindira kusingakoshi uye kuwedzeredza6.Naizvozvo, pane kudikanwa kwekugadzira ma biomarkers akavimbika uye anobatsira kuti aone nemazvo kenza yemapapu mumatanho ekutanga uye kusiyanisa akawanda mabhenekeri pakuona kwekutanga 7.
Kuongorora kwakadzama kwema molecular yeropa (serum, plasma, peripheral blood mononuclear cells), kusanganisira genomics, proteomics kana DNA methylation8,9,10, zvakonzera kufarira kukuru mukuwanikwa kwediagnostic biomarkers yekenza yemapapu.Zvichakadaro, nzira dzemetabolomics dzinoyera zvigadzirwa zvekupedzisira zvemagetsi zvinopesvedzerwa nemaitiro ekupedzisira uye exogenous zviito uye nekudaro zvinoshandiswa kufanotaura kutanga kwechirwere uye mhedzisiro.Liquid chromatography-tandem mass spectrometry (LC-MS) inzira inoshandiswa zvakanyanya yezvidzidzo zvemetabolomics nekuda kwekunzwa kwayo kukuru uye hukuru hwemasimba, hunogona kuvhara metabolites nemhando dzakasiyana dze physicochemical properties11,12,13.Kunyangwe ongororo yepasi rose yemetabolomic yeplasma/serum yakashandiswa kuzivisa biomarkers yakabatana nekenza yemapapu14,15,16,17 uye kugona kwekurapa,18 serum metabolite classifiers kusiyanisa pakati pehutsinye uye hutsinye hwemapapu manodule anoramba achidzidza zvakanyanya.-tsvakurudzo huru.
Adenocarcinoma uye squamous cell carcinoma ndiwo maviri makuru subtypes asiri diki cell kenza yemapapu (NSCLC).Yakasiyana-siyana CT yekuongorora bvunzo inoratidza kuti adenocarcinoma ndiyo inonyanya kuzivikanwa mhando yekenza yemapapu1,19,20,21.Muchidzidzo ichi, takashandisa ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) kuita ongororo yemetabolomics pahuwandu hwemasamples e695 serum, kusanganisira kutonga kwakanaka, benign pulmonary nodules, uye CT-yakaonekwa ≤3 cm.Kuongororwa kweStage I lung adenocarcinoma.Takaona pani ye serum metabolites inosiyanisa mapapu adenocarcinoma kubva kune benign nodules uye hutano kudzora.Nzira yekuvandudza ongororo yakaratidza kuti tryptophan isina kujairika uye glucose metabolism ndiyo yakajairika shanduko mumapapu adenocarcinoma kana ichienzaniswa nebenign nodules uye hutano kudzora.Chekupedzisira, isu takamisa uye takasimbisa serum metabolic classifier ine hunyanzvi hwepamusoro uye kunzwisiswa kusiyanisa pakati peakaipa uye benign pulmonary nodules akaonekwa neLDCT, iyo inogona kubatsira mukutanga kusiyanisa kuongororwa uye kuongororwa kwenjodzi.
Muchidzidzo chezvino, bonde- uye zera-rinofananidzwa serum samples dzakaunganidzwa retrospectively kubva 174 hutano kutonga, 292 varwere vane benign pulmonary nodules, uye 229 varwere vane stage I lung adenocarcinoma.Demographic maitiro ezvidzidzo zve695 anoratidzwa muSupplementary Table 1.
Sezvinoratidzwa muFigure 1a, huwandu hwe480 serum samples, kusanganisira 174 hutano hunodzora (HC), 170 benign nodules (BN), uye 136 nhanho I lung adenocarcinoma (LA) samples, dzakaunganidzwa paSun Yat-sen University Cancer Center.Discovery cohort yekusatariswa metabolomic profiling uchishandisa ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).Sezvinoratidzwa muSupplementary Figure 1, kusiyanisa metabolites pakati pe LA neHC, LA neBN zvakaonekwa kuti zvigadzire modhi yemhando uye nekuongororazve nzira yekusiyanisa nzira.104 samples dzakaunganidzwa neSun Yat-sen University Cancer Center uye 111 samples dzakaunganidzwa nezvimwe zvipatara zviviri zvakaiswa pasi pekusimbiswa kwemukati nekunze, zvichiteerana.
Chidzidzo chehuwandu muboka rekuwanikwa rakaitwa pasi rose serum metabolomics ongororo vachishandisa Ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).b Chikamu chidiki diki mativi erusarura ongororo (PLS-DA) yeyese metabolome ye480 serum samples kubva kune yekudzidza cohort, kusanganisira ine hutano kutonga (HC, n = 174), benign nodules (BN, n = 170), uye nhanho I lung adenocarcinoma. (Los Angeles, n = 136).+ESI, yakanaka electrospray ionization mode, -ESI, negative electrospray ionization mode.c–e Metabolites ane zvakawanda zvakasiyana zvakasiyana mumapoka maviri akapiwa (maviri-tailed Wilcoxon akasaina chiyero chebvunzo, chiyero chekuwanikwa chenhema chakagadziriswa p kukosha, FDR <0.05) inoratidzwa mutsvuku (peta shanduko> 1.2) uye yebhuruu (peta shanduko <0.83) .) inoratidzwa pamufananidzo wegomo rinoputika.f Hierarchical clustering heat mepu inoratidza mutsauko wakakura munhamba yeannotated metabolites pakati pe LA neBN.Source data inopiwa nenzira yemasource data mafaira.
Iyo yakazara serum metabolome ye174 HC, 170 BN uye 136 LA muboka rekuwanikwa yakaongororwa pachishandiswa UPLC-HRMS kuongorora.Isu tinotanga kuratidza kuti kutonga kwemhando yepamusoro (QC) masampula anobatana zvakasimba pakati peiyo isina kutariswa principal component analysis (PCA) modhi, ichisimbisa kugadzikana kwekuita kwechidzidzo chezvino (Supplementary Figure 2).
Sezvinoratidzwa mune chikamu chidiki-diki-rusarura ongororo (PLS-DA) muMufananidzo 1 b, takaona kuti pakanga paine misiyano yakajeka pakati peLA neBN, LA neHC mune yakanaka (+ESI) uye yakaipa (-ESI) electrospray ionization modes. .isolated.Nekudaro, hapana misiyano yakakosha yakawanikwa pakati peBN neHC mu +ESI uye -ESI mamiriro.
Takawana 382 zvakasiyana-siyana pakati pe LA neHC, 231 zvakasiyana-siyana pakati pe LA neBN, uye 95 yakasiyana-siyana pakati peBN neHC (Wilcoxon yakasaina rank test, FDR <0.05 uye kuchinja kwakawanda> 1.2 kana <0.83) (Mufananidzo .1c-e) ).Peaks yakawedzera kutsanangurwa (Supplementary Data 3) inopesana nedhatabhesi (mzCloud/HMDB/Chemspider raibhurari) nem/z kukosha, nguva yekuchengetedza uye kupatsanurwa kwemaspektrum kutsvaga (tsanangudzo inotsanangurwa muchikamu cheNzira) 22.Pakupedzisira, 33 uye 38 annotated metabolites ane misiyano yakakura muhuwandu yakaonekwa kune LA inopesana neBN (Mufananidzo 1f uye Supplementary Table 2) uye LA inopesana neHC (Supplementary Figure 3 uye Supplementary Table 2), maererano.Kusiyana neizvi, chete 3 metabolites ine misiyano yakakura mukuwanda yakaonekwa muBN uye HC (Supplementary Table 2), inopindirana nekupindirana pakati peBN neHC muPLS-DA.Aya mametabolites akasiyana-siyana anovhara zvakasiyana-siyana zvebiochemicals (Supplementary Figure 4).Zvakatorwa pamwechete, mhedzisiro iyi inoratidza shanduko huru muserum metabolome inoratidza kushanduka kwakashata kwekenza yemapapu yepakutanga kana ichienzaniswa nemanodules emapapu kana vanhu vane hutano.Zvichakadaro, kufanana kweserum metabolome yeBN neHC inoratidza kuti benign pulmonary nodules inogona kugovera maitiro akawanda ehupenyu nevanhu vane hutano.Tichifunga kuti epidermal growth factor receptor (EGFR) gene mutations inowanzoitika mumapapu adenocarcinoma subtype 23, takatsvaga kuona kukanganisa kwekuchinja kwemutyairi paserum metabolome.Takazoongorora iyo yakazara metabolomic profile ye72 kesi neEGFR chimiro mumapapu adenocarcinoma boka.Zvinofadza kuti takawana mapepa akafanana pakati peEGFR mutant varwere (n = 41) uye EGFR mhuka dzomusango (n = 31) muPCA kuongorora (Supplementary Figure 5a).Zvisinei, takaona 7 metabolites iyo yakawanda yakashandurwa zvakanyanya kune varwere vane EGFR mutation kana vachienzaniswa nevarwere vane wild-type EGFR (t test, p <0.05 uye fold change> 1.2 kana <0.83) (Supplementary Figure 5b).Ruzhinji rweaya metabolites (5 kubva pa7) ndeye acylcarnitines, iyo inoita basa rinokosha mumafuta acid oxidation nzira.
Sezvinoratidzwa mukufambiswa kwebasa kunoratidzwa muMufananidzo 2 a, ma biomarker ekuisa nhanho akatorwa pachishandiswa mashoma absolute shrinkage operators uye kusarudzwa kwakavakirwa pamakumi matatu nematatu akasiyana metabolites akaonekwa muLA (n = 136) uye BN (n = 170).Kunyanya kusanganiswa kwezvinosiyana (LASSO) - binary logistic regression model.Gumi-yakapetwa muchinjikwa-yakashandiswa kuedza kuvimbika kwemuenzaniso.Kusarudzika kusarudzwa uye paramende kugadzika kunogadziriswa nemukana wekuwedzera chirango chine parameter λ24.Global metabolomics analysis yakaitwazve yakazvimiririra mukusimbiswa kwemukati (n = 104) uye kunze kwekusimbisa (n = 111) mapoka kuti aedze maitiro ekugadzirisa kwemuenzaniso wekusarura.Somugumisiro, 27 metabolites mukutsvaga kwekutsvaga yakaonekwa seyakanakisisa yekusarura muenzaniso nehukuru hukuru hweAUC kukosha (Fig. 2b), pakati peiyo 9 yakanga yakawedzera basa uye 18 yakaderera basa muLA yakaenzaniswa neBN (Fig. 2c).
Kufamba kwebasa rekuvaka pulmonary nodule classifier, kusanganisira kusarudza iyo yakanakisa pani yeserum metabolites mune yekuwanikwa seti uchishandisa bhinary logistic regression modhi kuburikidza negumi-kupetwa-kuyambuka-kusimbisa uye kuongorora kufanotaura kuita mukati nekunze kweseti yekusimbisa.b Mchinji-kusimbisa nhamba dzeLASSO regression modhi ye metabolic biomarker kusarudzwa.Nhamba dzakapihwa pamusoro dzinomiririra avhareji yenhamba yezvinyorwa zvakasarudzwa pane yakapihwa λ.Mutsetse mutsvuku une doti unomiririra avhareji AUC kukosha kune inoenderana lambda.Grey kukanganisa mabhawa anomiririra hushoma uye hwakanyanya AUC kukosha.Mutsetse une doti unoratidza yakanakisa modhi ine makumi maviri nenomwe akasarudzwa biomarker.AUC, nzvimbo iri pasi peanogamuchira anoshanda maitiro (ROC) curve.c Peta shanduko dze27 dzakasarudzwa metabolites muboka reLA zvichienzaniswa neboka reBN riri muboka rekuwanikwa.Red column - activation.Koramu yebhuruu kuderera.d-f Receiver inoshanda hunhu (ROC) macurves anoratidza simba reiyo yekusarura modhi yakavakirwa pamakumi maviri nenomwe metabolite musanganiswa mukuwanikwa, mukati, uye kunze kwekusimbisa seti.Source data inopiwa nenzira yemasource data mafaira.
Muenzaniso wekufanotaura wakagadzirwa zvichienderana nekuremerwa kwekugadzirisa coefficients yeaya 27 metabolites (Supplementary Table 3).ROC kuongororwa kunobva pane idzi 27 metabolites yakapa nzvimbo pasi pe curve (AUC) kukosha kwe0.933, kuwanikwa kweboka rekunzwa kwaiva 0.868, uye chaiyo yaiva 0.859 (Fig. 2d).Zvichakadaro, pakati pe 38 annotated differential metabolites pakati LA neHC, seti yegumi nematanhatu metabolites yakawana AUC ye0.902 nekunzwa kwe0.801 uye chaiyo ye0.856 mukusarura LA kubva kuHC (Supplementary Figure 6a-c).AUC kukosha kwakavakirwa kwakasiyana kupeta shanduko zvikumbaridzo zvekusiyanisa metabolites zvakaenzaniswa zvakare.Takaona kuti mhando yemhando yakanyatsoita rusarura pakati peLA neBN (HC) apo kupeta shanduko level yakaiswa ku1.2 maringe ne1.5 kana 2.0 (Supplementary Figure 7a,b).Iyo yemhando yekuenzanisira, yakavakirwa pamapoka e27 metabolite, yakasimbiswazve mukati uye kunze cohorts.IAUC yaiva 0.915 (sensitivity 0.867, chaiyo 0.811) yekusimbisa mukati uye 0.945 (sensitivity 0.810, chaiyo 0.979) yekusimbisa kunze (Fig. 2e, f).Kuti uongorore kushanda kwe interlaboratory, 40 samples kubva kune yekunze cohort yakaongororwa murabhoritari yekunze sezvakatsanangurwa muchikamu cheNzira.Kurongeka kwakaringana kwakawana AUC ye0.925 (Supplementary Figure 8).Nekuti lung squamous cell carcinoma (LUSC) ndiyo yechipiri yakajairika subtype yeasiri diki cell kenza yemapapu (NSCLC) mushure memapapu adenocarcinoma (LUAD), isu takaedzawo yakasimbiswa inogona kushandiswa kwema metabolic profiles.BN uye 16 makesi eLUSC.Iyo AUC yerusarura pakati peLUSC neBN yaive 0.776 (Supplementary Figure 9), zvichiratidza kugona kushoma kana zvichienzaniswa nerusarura pakati peLUAD neBN.
Zvidzidzo zvakaratidza kuti ukuru hwemanodule pamifananidzo yeCT inopindirana zvakanaka nemukana wekurwara uye inoramba iri chirevo chikuru chekurapa nodule25,26,27.Kuongororwa kwedheta kubva kuboka guru rekuongorora kweNELSON kuongorora kwakaratidza kuti njodzi yezvakaipa mune zvidzidzo zvine nodes <5 mm yakanga yakafanana neyo mune zvidzidzo pasina nodes 28.Nokudaro, saizi shoma inoda kugara ichiongororwa CT i5 mm, sezvakakurudzirwa neBritish Thoracic Society (BTS), uye 6 mm, sezvakakurudzirwa neFleischner Society 29.Zvisinei, zvipembenene zvakakura kudarika 6 mm uye pasina maitiro akajeka akajeka, anonzi indeterminate pulmonary nodules (IPN), anoramba ari dambudziko guru mukuongorora uye kutungamira mune zvekliniki maitiro30,31.Isu takazoongorora kana saizi yenodule yakapesvedzera masiginecha emetabolomic tichishandisa masamples akabatanidzwa kubva mukuwanikwa uye emukati yekusimbisa cohorts.Tichitarisa pamakumi maviri nenomwe akasimbiswa biomarkers, takatanga taenzanisa maPCA profiles eHC uye BN sub-6 mm metabolomes.Takaona kuti dzakawanda data pfungwa nokuda HC uye BN akaturikidzana, zvichiratidza kuti serum metabolite mazinga akanga akafanana mumapoka maviri (Fig. 3a).Mamepu emhando dzakasiyana siyana saizi dzakaramba dzakachengeterwa muBN uye LA (Fig. 3b, c), nepo kupatsanurwa kwakaonekwa pakati peakaipa uye ane bhenen nodules mu 6-20 mm renji (Fig. 3d).Iri boka raive neAUC ye0.927, chaiyo ye0.868, uye kunzwisiswa kwe0.820 yekufanotaura kuipa kwemanodule anoyera 6 kusvika 20 mm (Fig. 3e, f).Mhedzisiro yedu inoratidza kuti iyo classifier inogona kubata shanduko yemetabolism yakakonzerwa neshanduko yekutanga yakaipa, zvisinei nehukuru hwemanodule.
ad Kuenzanisa kwePCA profiles pakati pemapoka akatsanangurwa zvichibva pane metabolic classifier ye27 metabolites.CC uye BN <6 mm.b BN <6 mm vs BN 6–20 mm.muLA 6–20 mm maringe neLA 20–30 mm.g BN 6-20 mm uye LA 6-20 mm.GC, n = 174;BN <6 mm, n = 153;BN 6–20 mm, n = 91;LA 6–20 mm, n = 89;LA 20–30 mm, n = 77. e Receiver operating character (ROC) curve inoratidza kusarura kwekuita kwemuenzaniso wemapundu 6–20 mm.f Hunhu hunogona kuverengerwa zvichienderana neiyo logistic regression modhi yemanodule anoyera 6-20 mm.Iyo grey ine dot line inomiririra yakaringana cutoff kukosha (0.455).Nhamba dziri pamusoro dzinomiririra chikamu chemhosva dzakarongwa kuLos Angeles.Shandisa t bvunzo yeMudzidzi ine miswe miviri.PCA, principal component analysis.AUC nzvimbo iri pasi pe curve.Source data inopiwa nenzira yemasource data mafaira.
Mienzaniso ina (yemakore 44-61 makore) ane mazamu akafanana emapapu (7-9 mm) akawedzerwa kusarudzwa kuti aratidze kushanda kweiyo yakarongwa yekufanotaura kwehuipi (Fig. 4a, b).Pakuongororwa kwekutanga, Nyaya yekutanga yakaratidzwa segunduru rakasimba rine calcification, chinhu chine chekuita nehunyoro, nepo Case 2 inoratidzwa sechinhu chisingaverengeki chakasimba chikamu chisina kujeka maficha.Mitatu mitatu yekutevera CT scans yakaratidza kuti nyaya idzi dzakaramba dzakagadzikana pamusoro pemakore e4 uye saka dzakaonekwa seine benign nodules (Fig. 4a).Kuenzaniswa nekiriniki yekuongorora ye serial CT scans, single-shot serum metabolite ongororo neiyo yazvino classifier modhi nekukurumidza uye nemazvo yakaratidza aya mabhenen nodules zvichibva pane probabilistic constraints (Tafura 1).Mufananidzo 4b muchiitiko che3 chinoratidza bhunu rine zviratidzo zvepleural retraction, iyo inowanzobatanidzwa nehuipi32.Mhosva yechina yakaunzwa sechinhu chisingaverengeki chakasimba zvishoma pasina humbowo hwechikonzero chakanaka.Mhosva idzi dzese dzakafanotaurwa dzakaipa zvichienderana neiyo classifier modhi (Tafura 1).Kuongororwa kwemapapu adenocarcinoma kwakaratidzwa nekuongorora kwe histopathological mushure mekuvhiyiwa kwemapapu (Fig. 4b).Kune yekunze yekusimbisa seti, iyo metabolic classifier yakanyatsofanotaura zviitiko zviviri zveindeterminate mapapu nodules akakura kupfuura 6 mm (Supplementary Figure 10).
CT mifananidzo yeaxial hwindo remapapu ezviitiko zviviri zvebhenign nodules.Kana 1, CT scan mushure memakore mana yakaratidza yakagadzikana yakasimba nodule inoyera 7 mm ine calcification muchikamu chekurudyi chezasi.Kana 2, CT scan mushure memakore mashanu yakaratidza kugadzikana, chikamu chakasimba chepfundo chine dhayamita 7 mm murobe rekurudyi repamusoro.b Axial hwindo CT mifananidzo yemapapu uye inoenderana pathological zvidzidzo zvezviitiko zviviri zvedanho I adenocarcinoma isati yaitwa mapapu.Mhosva yechitatu yakaburitsa nhanho ine dhayamita ye8 mm muchikamu chekurudyi chepamusoro chine pleural retraction.Mhosva yechina yakaratidza chikamu chakasimba chepasi-girazi nodule inoyera 9 mm kuruboshwe rwekumusoro lobe.Hematoxylin uye eosin (H&E) kusvibiswa kwetishu yemapapu yakadzokororwa (scale bar = 50 μm) inoratidza acinar yekukura pateni ye lung adenocarcinoma.Miseve inoratidza ma nodules akaonekwa pamifananidzo yeCT.H & E mifananidzo inomiririra mifananidzo yakawanda (> 3) microscopic minda inoongororwa nenyanzvi yezvirwere.
Kutorwa pamwe chete, mhedzisiro yedu inoratidza kukosha kunogona kuitika kweserum metabolite biomarkers mukusiyanisa kuongororwa kwepulmonary nodules, izvo zvinogona kuunza matambudziko pakuongororwa kweCT.
Zvichienderana neyakagadziriswa mutsauko wemetabolite pani, takatsvaga kuona biological correlates ekuchinja kukuru kwemetabolic.KEGG pathway enrichment analysis neMetaboAnalyst yakaratidza 6 yakajairika yakashandurwa zvakanyanya nzira pakati pemapoka maviri akapiwa (LA vs. HC uye LA vs. BN, yakagadziridzwa p ≤ 0.001, effect> 0.01).Kuchinja uku kwakaratidzwa nekuvhiringidzika mu pyruvate metabolism, tryptophan metabolism, niacin uye nicotinamide metabolism, glycolysis, TCA cycle, uye purine metabolism (Fig. 5a).Isu takazoenderera mberi nekuita zvakanangwa metabolomics kuona shanduko huru tichishandisa absolute quantification.Kutsunga kwemametabolites akajairwa munzira dzinowanzo chinjwa nekatatu quadrupole mass spectrometry (QQQ) uchishandisa echokwadi metabolite zviyero.Demographic characters of the metabolomics study target sample inowanikwa muSupplementary Table 4. Zvinoenderana nemigumisiro yedu yepasi rose yemetabolomics, kuongororwa kwehuwandu kwakasimbisa kuti hypoxanthine uye xanthine, pyruvate, uye lactate yakawedzerwa muLA yakaenzaniswa neBN uye HC (Fig. 5b, c, p <0.05).Nekudaro, hapana misiyano yakakosha mune aya metabolites yakawanikwa pakati peBN neHC.
KEGG nzira yekuvandudza ongororo yemametabolites akasiyana zvakanyanya muboka reLA zvichienzaniswa nemapoka eBN neHC.A two-tailed Globaltest yakashandiswa, uye p kukosha akagadziridzwa uchishandisa Holm-Bonferroni nzira (yakagadziridzwa p ≤ 0.001 uye mhedzisiro saizi> 0.01).b-d Violin zvirongwa zvinoratidza hypoxanthine, xanthine, lactate, pyruvate, uye tryptophan mazinga muserum HC, BN, uye LA yakatemwa neLC-MS/MS (n = 70 paboka).Mitsetse ine madota machena nemitema inoratidza pakati nepakati uye quartile, zvichiteerana.e Violin plot inoratidza yakajairwa Log2TPM (zvinyorwa pamiriyoni) mRNA kutaura kweSLC7A5 uye QPRT mumapapu adenocarcinoma (n = 513) zvichienzaniswa neyakajairika mapapu tishu (n = 59) muLUAD-TCGA dataset.Bhokisi jena rinomiririra interquartile renji, mutsara mutema wakatwasuka pakati unomiririra wepakati, uye mutsetse mutema wakatwasuka unobva mubhokisi unomiririra 95% yekuvimbana (CI).f Pearson correlation plot ye SLC7A5 uye GAPDH kutaura mu lung adenocarcinoma (n = 513) uye yakajairika mapapu tishu (n = 59) muTCGA dataset.Iyo grey nzvimbo inomiririra iyo 95% CI.r, Pearson coefficient yekubatanidza.g Normalized cellular tryptophan mazinga muA549 masero anotapurwa neasina specific shRNA control (NC) uye shSLC7A5 (Sh1, Sh2) yakatemwa neLC-MS/MS.Ongororo yenhamba yemasampuli mashanu akazvimirira ega muboka rega rega anoratidzwa.h Maserura mazinga eNADt (yakazara NAD, kusanganisira NAD+ neNADH) muA549 masero (NC) uye SLC7A5 kugogodza A549 masero (Sh1, Sh2).Ongororo yenhamba yemasampuli matatu akazvimirira ega muboka rega rega anoratidzwa.i Glycolytic chiitiko chemasero eA549 pamberi uye mushure meSLC7A5 kugogodza kwakayerwa neextracellular acidification rate (ECAR) (n = 4 biologically yakazvimirira samples paboka).2-DG, 2-deoxy-D-glucose.Mudzidzi ane miswe miviri t bvunzo yakashandiswa muna (b–h).Mu (g-i), mabhawa ekukanganisa anomiririra zvinoreva ± SD, kuedza kwega kwega kwakaitwa katatu zvakasununguka uye mhedzisiro yakafanana.Source data inopiwa nenzira yemasource data mafaira.
Tichifunga nezve kukanganiswa kwakakosha kwekushandurwa tryptophan metabolism muboka reLA, isu takaongororawo serum tryptophan mazinga muHC, BN, uye LA mapoka achishandisa QQQ.Takaona kuti serum tryptophan yakaderedzwa muLA kana ichienzaniswa neHC kana BN (p <0.001, Mufananidzo 5d), iyo inoenderana nezvakawanikwa zvekare kuti kutenderera tryptophan mazinga akadzikira muvarwere vane kenza yemapapu pane mune hutano kutonga kubva muboka rekutonga33,34. ,35.Imwe ongororo ichishandisa PET/CT tracer 11C-methyl-L-tryptophan yakawana kuti tryptophan chiratidzo chekuchengetedza nguva mukenza yemapapu yakawedzera zvakanyanya kana ichienzaniswa nemaronda asina kunaka kana akajairwa matishu36.Isu tinofungidzira kuti kudzikira kwe tryptophan muLA serum kunogona kuratidza inoshanda tryptophan kutorwa nemasero emukenza wemapapu.
Zvinozivikanwa zvakare kuti kuguma kwechigadzirwa chekynurenine nzira ye tryptophan catabolism ndeye NAD+37,38, inova yakakosha substrate yekuita kwe glyceraldehyde-3-phosphate ine 1,3-bisphosphoglycerate mu glycolysis39.Kunyange zvazvo zvidzidzo zvekare zvakatarisa pane basa re tryptophan catabolism mukudzivirira muviri, takatsvaga kujekesa kuwirirana pakati pe tryptophan dysregulation uye glycolytic nzira dzakaonekwa muchidzidzo chezvino.Solute transporter mhuri 7 nhengo 5 (SLC7A5) inozivikanwa kuve tryptophan transporter43,44,45.Quinolinic acid phosphoribosyltransferase (QPRT) ienzyme inowanikwa pasi penzira ye kynurenine inoshandura quinolinic acid kuNAMN46.Kuongororwa kweLUAD TCGA dataset yakaratidza kuti zvose SLC7A5 neQPRT zvakanyatsogadziriswa mumasumbu emamota zvichienzaniswa nenyama yakajairwa (Fig. 5e).Kuwedzera uku kwakaonekwa mumatanho I uye II pamwe chete nematanho III uye IV emapapu adenocarcinoma (Supplementary Figure 11), zvichiratidza kukanganisa kwepakutanga mu tryptophan metabolism inobatanidza ne tumorigenesis.
Pamusoro pezvo, iyo LUAD-TCGA dhata yakaratidza kuwirirana kwakanaka pakati peSLC7A5 neGAPDH mRNA kutaura mumasampuli evarwere vegomarara (r = 0.45, p = 1.55E-26, Mufananidzo 5f).Kusiyana neizvi, hapana kuwirirana kwakakosha kwakawanikwa pakati peakadaro masiginecha mumatishu emapapu akajairwa (r = 0.25, p = 0.06, Mufananidzo 5f).Knockdown yeSLC7A5 (Supplementary Figure 12) muA549 masero akaderedza zvakanyanya cellular tryptophan uye NAD (H) mazinga (Mufananidzo 5g, h), zvichiita kuti attenuated glycolytic basa sezvinoyerwa ne extracellular acidification rate (ECAR) (Mufananidzo 1).5i).Nekudaro, zvichibva pakuchinja kwemetabolism mu serum uye in vitro yekuona, isu tinofungidzira kuti tryptophan metabolism inogona kuburitsa NAD + kuburikidza nekynurenine nzira uye inotora basa rakakosha mukusimudzira glycolysis mukenza yemapapu.
Zvidzidzo zvakaratidza kuti nhamba yakawanda yezvisingaverengeki zvepulmonary nodules zvakaonekwa neLDCT zvinogona kutungamirira kune kudiwa kwekuwedzera kuongororwa kwakadai sePET-CT, lung biopsy, uye kunyanya kudarika nekuda kwenhema-positive kuongororwa kwechirwere.31 Sezvakaratidzwa mumufananidzo 6, chidzidzo chedu chakaratidza pani ye serum metabolites ine inogona kuongororwa kukosha iyo inogona kuvandudza njodzi stratification uye inotevera manejimendi yemapurmonary nodules akaonekwa neCT.
Pulmonary nodules inoongororwa uchishandisa yakaderera-dose computed tomography (LDCT) ine maficha ekufungidzira anoratidza bhenign kana zvakaipa zvikonzero.Mhedzisiro isina chokwadi yemanodule inogona kukonzera kushanya kwekutevera nguva nenguva, kupindira kusingakoshi, uye kunyanya kurapwa.Kuiswa kweserum metabolic classifiers ine diagnostic value inogona kuvandudza kuongororwa kwenjodzi uye kunotevera manejimendi emapulmonary nodules.PET positron emission tomography.
Dhata kubva kuUS NLST yekudzidza uye yeEuropean NELSON ongororo inoratidza kuti kuongorora mapoka ane njodzi yakakura ane yakaderera-dose computed tomography (LDCT) inogona kuderedza kufa kwekenza yemapapu1,3.Nekudaro, kuongororwa kwenjodzi uye kunotevera kwekiriniki manejimendi yenhamba huru yemapulmonary nodules akaonekwa neLDCT anoramba ari akaomarara.Chinangwa chikuru ndechekukwirisa kurongeka kwakaringana kweiyo iripo LDCT-based protocol nekubatanidza akavimbika biomarker.
Mamwe ma molecular biomarkers, akadai seropa metabolites, akaonekwa nekuenzanisa kenza yemapapu ine hutano kudzora15,17.Muchidzidzo chemazuva ano, takatarisa kushandiswa kweserum metabolomics kuongorora kusiyanisa pakati pehupamhi uye yakaipa pulmonary nodules yakaonekwa neLDCT.Isu takafananidza iyo yepasi rose serum metabolome yehutano kutonga (HC), benign lung nodules (BN), uye nhanho I lung adenocarcinoma (LA) samples tichishandisa UPLC-HRMS ongororo.Takaona kuti HC neBN vaive neakafanana metabolic profiles, nepo LA yakaratidza shanduko dzakakura zvichienzaniswa neHC neBN.Takaona seti mbiri dzeserum metabolites dzinosiyanisa LA kubva kuHC uye BN.
Ikozvino LDCT-based identification scheme yemanodule asina kunaka uye akaipa inonyanya kuenderana nehukuru, density, morphology uye kukura kwemanodule nekufamba kwenguva30.Zvidzidzo zvakapfuura zvakaratidza kuti saizi yemanodule ine hukama zvakanyanya neiyo mukana wekenza yemapapu.Kunyangwe muvarwere vane njodzi yakakura, njodzi yekushata mumanode <6 mm ndeye <1%.Ngozi yekurwara nemanodule anoyera 6 kusvika 20 mm inotangira pa8% kusvika 64%30.Naizvozvo, Fleischner Society inokurudzira cutoff dhayamita ye 6 mm yenguva dzose CT yekutevera.29 Zvisinei, kuongororwa kwengozi uye kutungamirirwa kweindeterminate pulmonary nodules (IPN) yakakura kudarika 6 mm haina kuitwa zvakakwana 31.Ikozvino manejimendi yecongenital heart disease inowanzobva pakurindira kumirira uye kazhinji CT monitoring.
Kubva pane yakasimbiswa metabolome, takaratidza kekutanga kupindirana kwemasaginecha emetabolomic pakati pevanhu vane hutano uye benign nodules <6 mm.Kufanana kwehupenyu kunopindirana nezvakaitika kare CT zvakawanikwa kuti njodzi yehutachiona hwemanodules <6 mm yakaderera seyezvidzidzo zvisina nodes.30 Zvinofanira kuonekwa kuti migumisiro yedu inoratidzawo kuti benign nodules <6 mm uye ≥6 mm ine yakakwirira. kufanana mumetabolomic profiles, zvichiratidza kuti tsanangudzo inoshanda yebenign etiology inopindirana pasinei nehukuru hwemanodule.Saka, yemazuva ano diagnostic serum metabolite panels inogona kupa imwechete assay sekutonga-kunze bvunzo apo nodules inotanga kuonekwa paCT uye inogona kuderedza serial monitoring.Panguva imwecheteyo, dhizaini rimwechete rema metabolic biomarkers raisiyanisa mavhuvhuvhu akashata ≥6 mm muhukuru kubva kune benign nodule uye akapa kufanotaura kwakarurama kweIPNs yehukuru hwakafanana uye ambiguous morphological features paCT mifananidzo.Iyi serum metabolism classifier yakaita zvakanaka mukufanotaura huipi hwemanodule ≥6 mm neAUC ye0.927.Kutorwa pamwe chete, mhedzisiro yedu inoratidza kuti yakasarudzika serum metabolomic siginecha inogona kunyatso ratidza yekutanga tumor-induced metabolic shanduko uye ine kukosha kwainogona senjodzi yekufanotaura, yakazvimiririra nekukura kwemanodule.
Zvikurukuru, lung adenocarcinoma (LUAD) uye squamous cell carcinoma (LUSC) ndiwo marudzi makuru easiri diki cell cancer cancer (NSCLC).Tichifunga kuti LUSC yakabatana zvakanyanya nekushandiswa kwefodya47 uye LUAD ndiyo inonyanya kuzivikanwa histology yemapundu emapapu akaonekwa paCT screening48, yedu classifier modhi yakavakirwa chaizvo nhanho I adenocarcinoma samples.Wang uye vaaishanda navo vakatarisawo paLUAD uye vakaratidza mapfumbamwe elipid siginecha vachishandisa lipidomics kusiyanisa kenza yemapapu ekutanga kubva kuvanhu vane hutano17.Isu takaedza yazvino classifier modhi pamakesi gumi nematanhatu edanho I LUSC uye makumi manomwe nemana mabhenen nodules uye takaona yakaderera LUSC kufanotaura chokwadi (AUC 0.776), zvichiratidza kuti LUAD neLUSC vangangove neyavo masiginecha emetabolomic.Chokwadi, LUAD neLUSC zvakaratidzwa kuti zvakasiyana mune etiology, biological mavambo uye genetic aberrations49.Naizvozvo, mamwe marudzi ehistology anofanirwa kuverengerwa mumhando dzekudzidzisa dzekuonekwa kwevanhu kenza yemapapu muzvirongwa zvekuongorora.
Pano, takaona nzira nhanhatu dzinowanzo chinjwa mumapapu adenocarcinoma kana zvichienzaniswa neane hutano kudzora uye benign nodules.Xanthine uye hypoxanthine zvakajairika metabolites ye purine metabolic nzira.Zvichienderana nemigumisiro yedu, vepakati vanobatanidzwa ne purine metabolism vakawedzera zvakanyanya mu serum kana matishu evarwere vane lung adenocarcinoma kana vachienzaniswa nekutonga kwakanaka kana varwere padanho rekutanga15,50.Yakakwira serum xanthine uye hypoxanthine mazinga anogona kuratidza anabolism inodiwa nekukurumidza kuwanda kwekenza maseru.Dysregulation yeglucose metabolism chiratidzo chinozivikanwa chekenza metabolism51.Pano, takaona kuwedzera kukuru kwepyruvate uye lactate muboka re LA zvichienzaniswa neHC neBN boka, iro rinoenderana nemishumo yapfuura yeglycolytic pathway abnormalities mu serum metabolome profiles yevasiri diki cell lung cancer (NSCLC) varwere uye hutano hunodzora.mhedzisiro inowirirana52,53.
Zvakakosha, takaona kuwirirana kwakasiyana pakati pepyruvate uye tryptophan metabolism muserum ye lung adenocarcinomas.Serum tryptophan mazinga akaderedzwa muboka reLA zvichienzaniswa neHC kana BN boka.Sezvineiwo, chidzidzo chekare chakakura chichishandisa boka rinotarisirwa rakaona kuti mazinga akaderera ekutenderera tryptophan akabatanidzwa nehuwandu hwekenza yemapapu 54.Tryptophan yakakosha amino acid yatinowana zvachose kubva muchikafu.Tinogumisa kuti serum tryptophan depletion mumapapu adenocarcinoma inogona kuratidza kukurumidza kupera kwemetabolite iyi.Zvinonyatsozivikanwa kuti chigadzirwa chekupedzisira che tryptophan catabolism kuburikidza ne kynurenine nzira ndiko kunobva de novo NAD + synthesis.Nekuti NAD + inogadzirwa zvakanyanya kuburikidza nenzira yekununura, kukosha kweNAD + mu tryptophan metabolism muhutano uye chirwere kunoramba kuchitemwa46.Ongororo yedu yedhatabhesi yeTCGA yakaratidza kuti kutaura kweye tryptophan transporter solute transporter 7A5 (SLC7A5) yakawedzera zvakanyanya mumapapu adenocarcinoma kana ichienzaniswa neyakajairwa kudzora uye yaienderana nekutaura kweiyo glycolytic enzyme GAPDH.Ongororo dzakapfuura dzakanyanya kutarisa nezve basa re tryptophan catabolism mukudzvanya antitumor immune response40,41,42.Pano tinoratidza kuti inhibition ye tryptophan uptake nekugogodza kweSLC7A5 mumasero emukenza wemapapu kunoguma nekudzikira kunotevera kwemasero eNAD mazinga uye kupindirana kwekuita kweglycolytic.Muchidimbu, chidzidzo chedu chinopa hwaro hwehupenyu hwekuchinja muserum metabolism ine chekuita nekuchinja kwakashata kwemapapu adenocarcinoma.
EGFR mutations ndiyo inonyanya kushandiswa kuchinja kwemutyairi muvarwere vane NSCLC.Muchidzidzo chedu, takaona kuti varwere vane EGFR mutation (n = 41) vaiva nehuwandu hwemetabolomic profiles yakafanana nevarwere vane wild-type EGFR (n = 31), kunyange zvazvo takawana kuderera kwemazinga e serum evamwe EGFR mutant varwere mu acylcarnitine varwere.Basa rakagadziriswa re acylcarnitines nderokutakura mapoka e-acyl kubva ku cytoplasm kusvika kune mitochondrial matrix, zvichiita kuti oxidation yemafuta acids ibudise simba 55.Zvichienderana nezvatakawana, ongororo ichangoburwa yakaratidzawo akafanana metabolome profiles pakati peEGFR mutant uye EGFR mhuka dzesango nekuongorora metabolome yepasi rose ye102 lung adenocarcinoma tissue samples50.Sezvineiwo, acylcarnitine yemukati yakawanikwa zvakare muEGFR mutant boka.Nokudaro, kana kuchinja kwemazinga e-acylcarnitine kunoratidza EGFR-induced metabolic shanduko uye nzira dziri pasi pema molecular dzinogona kukodzera kuwedzera kudzidza.
Mukupedzisa, chidzidzo chedu chinogadza serum metabolic classifier yekusiyanisa kuongororwa kwepulmonary nodules uye inopa mafambiro ebasa anogona kukwirisa kuongororwa kwenjodzi uye kufambisa manejimendi ekiriniki zvichienderana neCT scan yekuongorora.
Ichi chidzidzo chakabvumidzwa neEthics Committee yeSun Yat-sen University Cancer Hospital, Yekutanga Yakabatana Chipatara cheSun Yat-sen University, uye Ethics Committee yeZhengzhou University Cancer Hospital.Mumapoka ekuwanikwa uye emukati ekusimbisa, 174 sera kubva kuvanhu vane hutano uye 244 sera kubva kune benign nodules yakaunganidzwa kubva kune vanhu vari kuongororwa gore negore kuDhipatimendi reCancer Control and Prevention, Sun Yat-sen University Cancer Center, uye 166 benign nodules.serum.Stage I lung adenocarcinomas yakaunganidzwa kubva kuSun Yat-sen University Cancer Center.Muboka rekusimbisa rekunze, maive nemakesi makumi mana nemasere emawere asina kunaka, makumi matatu nepfumbamwe enhanho I lung adenocarcinoma kubva kuFirst Affiliated Hospital yeSun Yat-sen University, uye makumi maviri nemana echikamu I lung adenocarcinoma kubva kuZhengzhou Cancer Hospital.Sun Yat-sen University Cancer Center yakaunganidzawo nyaya gumi nenhanhatu dzepastage I squamous cell cancer yemapapu kuyedza kugona kweiyo yakagadziriswa metabolic classifier (maitiro emurwere anoratidzwa muSupplementary Table 5).Mienzaniso kubva kuboka rezviwanikwa uye boka rekuongorora remukati rakaunganidzwa pakati paNdira 2018 naChivabvu 2020. Mienzaniso yeboka rekuongorora rekunze yakaunganidzwa pakati paNyamavhuvhu 2021 naGumiguru 2022. Kuti kuderedze rusarura, nhamba dzinoenzana dzenyaya dzevarume nevakadzi dzakapihwa kune imwe neimwe. cohort.Discovery Team uye Yemukati Yekuongorora Chikwata.Mutori wechikamu murume kana mukadzi akatemerwa zvichibva pakuzvizivisa.Mvumo ine ruzivo yakawanikwa kubva kune vese vatori vechikamu uye hapana muripo wakapihwa.Zvidzidzo zvine benign nodules vaive avo vane yakagadzikana CT scan mamakisi pa2 kusvika ku5 makore panguva yekuongororwa, kunze kwe1 kesi kubva kune yekunze yekusimbisa sampuli, iyo yakaunganidzwa preoperative uye yakaonekwa nehistopathology.Kunze kwechirwere chebronchitis chisingaperi.Mhosva dzeLung adenocarcinoma dzakaunganidzwa kusati kwaitwa mapapu uye kusimbiswa nekuongororwa kwepathological.Kutsanya ropa samples dzakaunganidzwa mu serum separation chubhu pasina chero anticoagulants.Ropa sampuli dzakagwamba kweawa imwe patembiricha yekamuri uyezve centrifuged pa2851 × g kwemaminetsi gumi pa4 ° C kuunganidza serum supernatant.Serum aliquots yakaomeswa nechando pa -80 ° C kusvika metabolite yatorwa.Dhipatimendi rekudzivirira kenza uye kuongororwa kwekurapa kweZuva Yat-sen University Cancer Center yakaunganidza dziva reserum kubva kune zana vanopa vane hutano, kusanganisira nhamba yakaenzana yevarume nevakadzi vane makore makumi mana kusvika makumi mashanu nemashanu.Mavhoriyamu akaenzana ega ega evanopa sampu akasanganiswa, dziva rakaguma rakatorwa uye rakachengetwa pa -80 ° C.Musanganiswa weserum wakashandiswa sereferensi yezvinhu zvekutonga kwemhando uye kumisa data.
Reference serum uye test samples dzakanyungudutswa uye metabolites dzakatorwa uchishandisa nzira yakasanganiswa yekubvisa (MTBE / methanol / mvura) 56.Muchidimbu, 50 μl ye serum yakasanganiswa ne225 μl yeaizi-inotonhora methanol uye 750 μl yechando-inotonhora methyl tert-butyl ether (MTBE).Kurudzira musanganiswa uye incubate paaizi kweawa imwe.Iwo masampuli akabva asanganiswa uye vortex yakasanganiswa ne188 μl yeMS-giredhi mvura ine mukati memazinga (13C-lactate, 13C3-pyruvate, 13C-methionine, uye 13C6-isoleucine, yakatengwa kubva kuCambridge Isotope Laboratories).Musanganiswa wakabva waiswa centrifuged pa 15,000 × g ye 10 min pa 4 ° C, uye chikamu chepasi chakaendeswa mumachubhu maviri (125 μL imwe neimwe) yeLC-MS kuongororwa mune zvakanaka uye zvisina kunaka modes.Pakupedzisira, sampuli yakanyungudutswa kusvika pakuoma mune yakakwirira-kumhanya vacuum concentrator.
Iyo yakaomeswa metabolites yakagadziridzwa zvakare mu 120 μl ye80% acetonitrile, yakavharidzirwa kwemaminitsi mashanu, uye centrifuged pa 15,000 × g ye 10 min pa 4 ° C.Masupernatants akatamisirwa muamber girazi mabhodhoro ane microinsets yezvidzidzo zvemetabolomics.Kusatariswa kwemetabolomics kuongororwa pane yekupedzisira-inoshanda mvura chromatography-yakakwirira-resolution mass spectrometry (UPLC-HRMS) chikuva.Metabolites yakaparadzaniswa uchishandisa Dionex Ultimate 3000 UPLC system uye ACQUITY BEH Amide column (2.1 × 100 mm, 1.7 μm, Mvura).Mune yakanaka ion mode, nharembozha dzaive 95% (A) uye 50% acetonitrile (B), imwe neimwe iine 10 mmol/L ammonium acetate uye 0.1% formic acid.Mumamiriro ezvinhu asina kunaka, zvikamu zvefoni A uye B zvine 95% uye 50% acetonitrile, zvichienderana, zvikamu zvose zvine 10 mmol / L ammonium acetate, pH = 9. Purogiramu ye gradient yaiva inotevera: 0-0.5 min, 2% B;0.5–12 min, 2–50% B;12–14 min, 50–98% B;14–16 min, 98% B;16–16.1.min, 98 –2% B;16.1-20 min, 2% B. Mutsara wakachengetwa pa40 ° C uye sampuli pa10 ° C mu autosampler.Chiyero chekuyerera chaive 0.3 ml / min, iyo jekiseni vhoriyamu yaive 3 μl.A Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) ine electrospray ionization (ESI) sosi yaishandiswa in full scan mode uye yakabatanidzwa neddMS2 monitoring mode kuunganidza mavhoriyamu makuru edata.The MS parameters akaiswa sezvinotevera: spray voltage +3.8 kV/- 3.2 kV, capillary tembiricha 320°C, kudzivirira gasi 40 arb, ebetsero gasi 10 arb, probe heater tembiricha 350°C, scanning range 70-1050 m/h, resolution.70 000. Data yakawanikwa uchishandisa Xcalibur 4.1 (Thermo Fisher Scientific).
Kuongorora mhando yedata, pooled quality control (QC) samples dzakagadzirwa nekubvisa 10 μL aliquots eiyo supernatant kubva kumuenzaniso wega wega.Manhanhatu emhando yekudzora majekiseni emuenzaniso akaongororwa pakutanga kwekutevedzana kwekuongorora kuongorora kugadzikana kweUPLC-MS system.Quality control samples dzinobva dzaunzwa nguva nenguva mubatch.Ese mabheji gumi nerimwe emasampuli e serum muchidzidzo ichi akaongororwa neLC-MS.Aliquots yemusanganiswa we serum pool kubva kune zana vane hutano vanopa vakashandiswa sechinhu chereferensi mumabheji akateerana kutarisa maitiro ekubvisa uye kugadzirisa mhedzisiro yebatch-to-batch.Kusatariswa kwemetabolomics kuongororwa kwechikwata chekuwana, chikwata chekusimbisa mukati, uye kunze kwekusimbisa chikwata chakaitwa paMetabolomics Center yeSun Yat-sen University.Rabhoritari yekunze yeGuangdong University of Technology Analysis uye Testing Center yakaongororawo masample makumi mana kubva kuboka rekunze kuyedza mashandiro emhando yeclassifier.
Mushure mekubviswa nekugadziriswa, huwandu hwakakwana hweserum metabolites hwakayerwa uchishandisa ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) ine electrospray ionization (ESI) sosi mune akawanda maitiro ekutarisa (MRM) maitiro.An ACQUITY BEH Amide column (2.1 × 100 mm, 1.7 μm, Mvura) yakashandiswa kuparadzanisa metabolites.Nharembozha yaisanganisira 90% (A) uye 5% acetonitrile (B) ine 10 mmol/L ammonium acetate uye 0.1% ammonia mhinduro.Chirongwa chegradient chaive seizvi: 0–1.5 min, 0% B;1.5–6.5 min, 0–15% B;6.5–8 min, 15% B;8–8.5 min, 15%–0% B;8.5–11.5 min, 0%B.Iyo column yakachengetedzwa pa40 ° C uye sampuli pa10 ° C mune autosampler.Chiyero chekuyerera chaive 0.3 mL / min uye vhoriyamu yejekiseni yaive 1 μL.MS parameters yakaiswa sezvinotevera: capillary voltage ± 3.5 kV, nebulizer pressure 35 psi, sheath gas flow 12 L/min, sheath gas tembiricha 350 ° C, kuomesa gasi tembiricha 250 ° C, uye kuomesa gasi kuyerera 14 l/min.Kushandurwa kweMRM kwe tryptophan, pyruvate, lactate, hypoxanthine uye xanthine vaiva 205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 uye 151.0–107.9 zvichiteerana.Data yakaunganidzwa pachishandiswa Mass Hunter B.07.00 (Agilent Technologies).Kune serum samples, tryptophan, pyruvate, lactate, hypoxanthine, uye xanthine zvakaverengerwa pachishandiswa calibration curves yeyakajairwa musanganiswa mhinduro.Kune maseru sampuli, tryptophan yemukati yakajairwa kune yemukati mwero uye sero mapuroteni mashoma.
Peak extraction (m/z uye nguva yekuchengeta (RT)) yakaitwa pachishandiswa Compound Discovery 3.1 uye TraceFinder 4.0 (Thermo Fisher Scientific).Kubvisa misiyano inogona kuitika pakati pemabhechi, peak yega yega yemuenzaniso webvunzo yakakamurwa nehunhu hwepamusoro hwechinhu chereferenzi kubva kubhechi rimwe chete kuti vawane kuwanda.Iko kuenzaniswa kwemaitiro ekutsauka kwemaitiro emukati zvisati zvaitika uye shure kwekugadziriswa kunoratidzwa muSupplementary Table 6. Kusiyana pakati pemapoka maviri aya kwakaratidzwa nekunyepa kwekutsvaga kwekutsvaga (FDR <0.05, Wilcoxon yakasaina rank test) uye kuchinja kwepeta (> 1.2 kana <0.83).Raw MS data yezvakabviswa uye referenzi serum-yakagadziriswa MS data inoratidzwa muSupplementary Data 1 uye Supplementary Data 2, zvichiteerana.Peak annotation yakaitwa yakavakirwa pamatanho mana akatsanangurwa ekuzivikanwa, kusanganisira akacherechedzwa metabolites, putatively annotated compounds, putatively characterized makomisheni makirasi, uye asingazivikanwi makomisheni 22.Zvichienderana nekutsvaga kwedatabase muCompound Discovery 3.1 (mzCloud, HMDB, Chemspider), biological compounds ine MS/MS inofananidzwa yakasimbiswa miyero kana chaiwo matchanotsidzo mumzCloud (score> 85) kana Chemspider akazosarudzwa sepakati pakati pemetabolome yekusiyanisa.Peak annotations yechinhu chimwe nechimwe inosanganisirwa muSupplementary Data 3. MetaboAnalyst 5.0 yakashandiswa kuongororwa univariate ye sum-normalized metabolite abundance.MetaboAnalyst 5.0 yakaongororawo KEGG nzira yekuvandudza ongororo yakavakirwa pamatabolites akasiyana zvakanyanya.Principal component analysis (PCA) uye partial less squares discriminant analysis (PLS-DA) yakaongororwa pachishandiswa ropls software package (v.1.26.4) ine stack normalization uye autoscaling.The optimal metabolite biomarker modhi yokufanotaura nodule kushata akagadzirwa kushandisa binary rogistic regression zvishoma mhedziso shrinkage uye kusarudza opareta (LASSO, R pasuru v.4.1-3).Kuitwa kwemuenzaniso werusarura mukutsvaga uye kugadzirisa zvigadziro zvakaratidzwa nekufungidzira AUC inobva paROC kuongororwa maererano nepROC package (v.1.18.0.).Iyo yakanyanya kunaka mukana cutoff yakawanikwa zvichibva pane yakanyanya Youden index yemuenzaniso (sensitivity + specificity - 1).Masamples ane mashoma kana makuru pane chikumbaridzo anozofanotaurwa sebenign nodules uye lung adenocarcinoma, zvichiteerana.
A549 maseru (#CCL-185, American Type Culture Collection) akakurira muF-12K yepakati ine 10% FBS.Short hairpin RNA (shRNA) sequences yakananga SLC7A5 uye nontargeting control (NC) yakaiswa mu lentiviral vector pLKO.1-puro.Iyo antisense sequences ye shSLC7A5 inotevera: Sh1 (5'-GGAGAAACCTGATGAACAGTT-3′), Sh2 (5'-GCCGTGGACTTCGGGAACTAT-3′).Masoja ekudzivirira chirwere kuSLC7A5 (#5347) uye tubulin (#2148) akatengwa kubva kuCell Signaling Technology.Masoja ekudzivirira chirwere kuSLC7A5 uye tubulin akashandiswa pakuderedzwa kwe1: 1000 yeWestern blot analysis.
Iyo Seahorse XF Glycolytic Stress Test inoyera extracellular acidification (ECAR) mazinga.Muchiyedzo, glucose, oligomycin A, uye 2-DG zvakapihwa zvakatevedzana kuyedza cellular glycolytic kugona sekuyerwa neECAR.
Masero eA549 akatapurwa neasina-targeting control (NC) uye shSLC7A5 (Sh1, Sh2) akaputirwa usiku humwe mu10 cm dhayamita ndiro.Sero metabolites yakatorwa ne 1 ml yechando-inotonhora 80% aqueous methanol.Masero ari mumushonga wemethanol akakweshwa, akaunganidzwa muchubhu itsva, uye akaiswa centrifuged pa15,000 × g kwemaminetsi gumi nemashanu pa4°C.Unganidza 800 µl ezvemhando yepamusoro uye woomesa uchishandisa high-speed vacuum concentrator.Iyo yakaomeswa metabolite pellets yakabva yaongororwa mazinga etryptophan achishandisa LC-MS/MS sezvinotsanangurwa pamusoro apa.Cellular NAD(H) mazinga muA549 maseru (NC uye shSLC7A5) akayerwa pachishandiswa huwandu hweNAD+/NADH colorimetric kit (#K337, BioVision) zvinoenderana nemirairo yemugadziri.Mapuroteni mazinga akayerwa kune yega yega sampu kuti ajaire huwandu hwemetabolites.
Hapana nzira dzenhamba dzakashandiswa kutanga kuona saizi yemuenzaniso.Zvidzidzo zvekare zvemetabolomics zvine chinangwa chekuwanikwa kwebiomarker15,18 zvakaonekwa semabhenji ekumisikidza saizi, uye zvichienzaniswa nemishumo iyi, sampuro yedu yaive yakakwana.Hapana sampuli dzakabviswa kubva kuboka rezvidzidzo.Serum samples dzakagoverwa kuboka rekutsvaga (306 kesi, 74.6%) uye boka remukati rekusimbisa (104 kesi, 25.4%) yezvidzidzo zvisingatarisirwi zvemetabolomics.Isu takasarudzawo zvisina tsarukano zviitiko zve70 kubva kuboka rega rega kubva pakuwanikwa kwakaiswa kune zvakanangwa zvidzidzo zvemetabolomics.Vaongorori vakapofumadzwa pakugoverwa kweboka panguva yeLC-MS yekuunganidza data uye kuongororwa.Statistical ongororo yemetabolomics data uye maseru kuyedza zvinotsanangurwa mune zvakateerana Mhedzisiro, Figure Ngano, uye Nzira zvikamu.Quantification yemaserura tryptophan, NADT, uye glycolytic chiitiko chakaitwa katatu zvakasununguka nemhedzisiro yakafanana.
Kuti uwane rumwe ruzivo nezve dhizaini yekudzidza, ona Natural Portfolio Report Abstract ine chekuita nechinyorwa ichi.
Iyo mbishi MS data yezvinhu zvakabviswa uye yakajairwa MS data yereferenzi serum inoratidzwa muSupplementary Data 1 uye Supplementary Data 2, zvichiteerana.Peak annotations for different features inoratidzwa muSupplementary Data 3. Iyo LUAD TCGA dataset inogona kutorwa kubva https://portal.gdc.cancer.gov/.Iyo data yekuisa yekuronga girafu inopihwa mune iyo source data.Source data inopihwa pachinyorwa ichi.
National Lung Screening Study Group, nezvimwewo.Northern England.J. Med.365, 395–409 (2011).
Kramer, BS, Berg, KD, Aberle, DR uye Muporofita, PC Kuongororwa kenza yemapapu uchishandisa yakaderera-dose helical CT: mhedzisiro kubva kuNational Lung Screening Study (NLST).J. Med.Screen 18, 109–111 (2011).
De Koning, HJ, nevamwe.Kuderedza kufa kwekenza yemapapu nevolumetric CT yekuongorora muyedzo isina kujairika.Northern England.J. Med.382, 503–513 (2020).
Nguva yekutumira: Sep-18-2023