Kuongororwa kwakasiyana kwemapundu emapapu kwakaonekwa ne computed tomography (CT) kuchiri dambudziko mukurapa. Pano, tinotsanangura metabolome yepasi rose yemasampuli mazana mana nemakumi masere emuropa, kusanganisira ma controls ane hutano, ma pundu emapapu asina hutano, uye stage I lung adenocarcinoma. Adenocarcinomas inoratidza ma profiles akasiyana e metabolomic, nepo ma pundu emapapu asina hutano nevanhu vane hutano vane kufanana kwakanyanya muma profiles e metabolomic. Muboka rekuwana (n = 306), seti yema metabolites makumi maviri nemanomwe akaonekwa kuti asiyanise pakati pema pundu emapapu asina hutano neakaipa. AUC yemuenzaniso we discriminant mu internal validation (n = 104) uye external validation (n = 111) mapoka aive 0.915 na 0.945, zvichiteerana. Kuongororwa kwenzira kwakaratidza kuwedzera kwe glycolytic metabolites kwakabatana nekudzikira kwe tryptophan mu lung adenocarcinoma serum zvichienzaniswa nema pundu emapapu asina hutano uye ma controls ane hutano, uye zvakaratidza kuti tryptophan uptake inokurudzira glycolysis mumasero ekenza yemapapu. Chidzidzo chedu chinosimbisa kukosha kwezvibodzwa zve serum metabolite mukuongorora njodzi yemapundu emapapu akaonekwa neCT.
Kuongororwa kwechirwere nekukurumidza kwakakosha kuti pave nekuwedzera mwero wekurarama kwevarwere vegomarara. Zvakabuda muUS National Lung Cancer Screening Trial (NLST) neEuropean NELSON Study zvakaratidza kuti kuongororwa ne computed tomography (LDCT) ine dose shoma kunogona kuderedza zvakanyanya kufa kwegomarara remapapu mumapoka ane njodzi huru1,2,3. Kubva pakushandiswa kwakawanda kweLDCT pakuongorora gomarara remapapu, kuwanda kwezviratidzo zvemapundu emapapu asina zviratidzo kwakaramba kuchiwedzera 4. Mapundu emapapu anotsanangurwa se focal opacities anosvika 3 cm mudhayamita 5. Tinosangana nematambudziko pakuongorora mukana wegomarara uye kubata nehuwandu hwakawanda hwemapundu emapapu akaonekwa netsaona paLDCT. Kusakwana kweCT kunogona kutungamira mukuongororwa nguva nenguva uye mhedzisiro yenhema, zvichitungamira mukupindira kusingakoshi uye kurapwa zvakanyanya6. Nokudaro, pane chikonzero chekugadzira biomarkers dzakavimbika uye dzinobatsira kuti vaone kenza yemapapu nemazvo mumatanho ekutanga uye kusiyanisa mapundu mazhinji asina njodzi pakuonekwa kwekutanga 7.
Kuongororwa kwakazara kwemasero eropa (serum, plasma, masero eropa eropa re peripheral), kusanganisira genomics, proteomics kana DNA methylation8,9,10, kwakaita kuti pave nekufarira kukuru mukuwana zviratidzo zvekuongorora kenza yemapapu. Panguva iyi, nzira dze metabolomics dzinoyera zvigadzirwa zvemasero zvinopesvedzerwa nezviito zve endogenous uye exogenous uye nekudaro dzinoshandiswa kufanotaura kutanga kwechirwere uye mhedzisiro yacho. Liquid chromatography-tandem mass spectrometry (LC-MS) inzira inoshandiswa zvakanyanya muzvidzidzo zve metabolomics nekuda kwekunzwisisa kwayo kwakanyanya uye nzvimbo yakakura ye dynamic, iyo inogona kufukidza metabolites dzine hunhu hwakasiyana hwe physicochemical11,12,13. Kunyangwe kuongorora metabolomic kwepasi rose kweplasma/serum kwakashandiswa kuona biomarkers dzine chekuita nekuongorora kenza yemapapu14,15,16,17 uye kushanda zvakanaka kwekurapa,18 serum metabolite classifiers yekusiyanisa pakati pema nodules emapapu asina njodzi uye ane malignant zvichiri kudzidzwa zvakanyanya. -tsvakurudzo huru.
Adenocarcinoma nesquamous cell carcinoma ndidzo mhando mbiri huru dzekenza yemapapu isiri diki (NSCLC). Kuongororwa kwakasiyana-siyana kweCT kunoratidza kuti adenocarcinoma ndiyo mhando inonyanya kuzivikanwa yekenza yemapapu1,19,20,21. Muchidzidzo ichi, takashandisa ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) kuongorora metabolomics pane huwandu hwe695 serum samples, kusanganisira healthy controls, benign pulmonary nodules, uye CT-detected ≤3 cm. Kuongorora Stage I lung adenocarcinoma. Takaona panel yema serum metabolites anosiyanisa lung adenocarcinoma kubva kune benign nodules uye healthy controls. Pathway enrichment analysis yakaratidza kuti abnormal tryptophan uye glucose metabolism ishanduko dzakajairika mu lung adenocarcinoma zvichienzaniswa ne benign nodules uye healthy controls. Pakupedzisira, takagadzira uye takasimbisa musanganiswa we serum metabolic classifier une hunyanzvi hwakanyanya uye hunhu hwekusiyanisa pakati pemapundu ane kenza uye asina njodzi anowanikwa neLDCT, izvo zvinogona kubatsira mukuongororwa kwekutanga kwakasiyana uye kuongororwa kwenjodzi.
Muchidzidzo chazvino, sampuro dzeropa dzinoenderana nemurume nemukadzi nezera dzakatorwa kubva kuvanhu 174 vane hutano hwakanaka, varwere 292 vane maronda emapapu asina njodzi, uye varwere 229 vane chirwere chemapapu chedanho rekutanga. Hunhu hwevanhu 695 vakaongororwa hunoratidzwa muSupplementary Table 1.
Sezvakaratidzwa muMufananidzo 1a, sampuli dzemuropa dzinosvika 480, dzinosanganisira 174 healthy control (HC), 170 benign nodules (BN), uye 136 stage I lung adenocarcinoma (LA), dzakatorwa paSun Yat-sen University Cancer Center. Kuwanikwa kweboka rekuongorora metabolomic isina kunangwa uchishandisa ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Sezvakaratidzwa muMufananidzo 1 wekuwedzera, differential metabolites pakati peLA neHC, LA neBN dzakaonekwa kuti dzigadzire classification model uye dziongorore differential pathway analysis. 104 sampuro dzakatorwa neSun Yat-sen University Cancer Center uye 111 sampuro dzakatorwa nedzimwe zvipatara zviviri dzakaongororwa mukati nekunze, zvichiteerana.
a Vanhu vakaongororwa muboka rekutsvaga vakaongororwa neropa rese vachishandisa ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). b Kuongororwa kwechikamu chechikamu chechikamu chechikamu chechikamu (PLS-DA) chemetabolome yese ye480 serum samples kubva kuboka rekudzidza, kusanganisira healthy controls (HC, n = 174), benign nodules (BN, n = 170), uye stage I lung adenocarcinoma (Los Angeles, n = 136). +ESI, positive electrospray ionization mode, -ESI, negative electrospray ionization mode. c–e Metabolites ane huwandu hwakasiyana mumapoka maviri akapihwa (two-tailed Wilcoxon signed rank test, false discovery rate adjusted p value, FDR <0.05) anoratidzwa mutsvuku (fold change > 1.2) uye bhuruu (fold change < 0.83). ) anoratidzwa pamufananidzo wevolcano. f Mepu yekupisa yekubatanidza mapoinzi inoratidza mutsauko mukuru muhuwandu hwemametabolites ane ruzivo pakati peLA neBN. Data rekwakabva rinopihwa muchimiro chemafaira edata rekwakabva.
Metabolome yese yeropa ye174 HC, 170 BN uye 136 LA muboka rekutsvaga yakaongororwa uchishandisa ongororo yeUPLC-HRMS. Kutanga tinoratidza kuti sampuro dzekutonga mhando (QC) dzakaungana zvakasimba pakati pemuenzaniso wekuongorora kwechikamu chikuru chisina kutarisirwa (PCA), zvichisimbisa kugadzikana kwekushanda kwechidzidzo ichi (Mufananidzo Wekuwedzera 2).
Sezvakaratidzwa muchikamu chekuongorora chechikamu chechikamu chechikamu chechikamu chechikamu (PLS-DA) muMufananidzo 1 b, takaona kuti paive nemusiyano wakajeka pakati peLA neBN, LA neHC mu positive (+ESI) uye negative (−ESI) electrospray ionization modes. dzakatsaurwa. Zvisinei, hapana mutsauko wakakosha wakawanikwa pakati peBN neHC mu +ESI ne -ESI mamiriro.
Takawana zvinhu zvakasiyana-siyana 382 pakati peLA neHC, zvinhu zvakasiyana-siyana 231 pakati peLA neBN, uye zvinhu zvakasiyana-siyana 95 pakati peBN neHC (Wilcoxon signed rank test, FDR <0.05 uye multiple change >1.2 kana <0.83) (Mufananidzo .1c-e). . Mapeaks akawedzerwa kunyorwa (Supplementary Data 3) zvichienderana nedatabase (mzCloud/HMDB/Chemspider library) ne m/z value, retention time uye fragmentation mass spectrum search (tsananguro dzakatsanangurwa muchikamu cheMethods) 22. Pakupedzisira, metabolites 33 ne38 dzakanyorwa dzine musiyano mukuru muhuwandu dzakaonekwa dzeLA vs BN (Mufananidzo 1f neSupplementary Table 2) uye LA vs HC (Supplementary Picture 3 neSupplementary Table 2), zvichiteerana. Kusiyana neizvi, ma metabolites matatu chete ane mutsauko mukuru muhuwandu akaonekwa muBN neHC (Supplementary Table 2), zvichienderana nekupindirana kuripo pakati peBN neHC muPLS-DA. Ma metabolites aya akasiyana anofukidza mhando dzakasiyana dzemakemikari (Supplementary Picture 4). Zvichitorwa pamwe chete, mhedzisiro iyi inoratidza shanduko huru mu serum metabolome inoratidza shanduko yakaipa yekenza yemapapu yekutanga kana tichienzanisa nema nodules emapapu asina njodzi kana vanhu vane hutano. Panguva imwecheteyo, kufanana kwe serum metabolome yeBN neHC kunoratidza kuti ma nodules asina njodzi anogona kugovana hunhu hwakawanda hwezvipenyu nevanhu vane hutano. Tichifunga kuti epidermal growth factor receptor (EGFR) gene mutations inowanzoonekwa mu lung adenocarcinoma subtype 23, takatsvaga kuona kuti driver mutations inokanganisa sei serum metabolome. Takazoongorora metabolomic profile yese yezviitiko makumi manomwe nembiri zvine EGFR status mu lung adenocarcinoma group. Zvinofadza kuti, takawana ma profile akafanana pakati pevarwere vane EGFR mutant (n = 41) nevarwere vane EGFR wild-type (n = 31) mu PCA analysis (Supplementary Picture 5a). Zvisinei, takaona metabolites 7 dzine huwandu hwakachinjwa zvakanyanya muvarwere vane EGFR mutation zvichienzaniswa nevarwere vane wild-type EGFR (t test, p < 0.05 uye fold change > 1.2 kana < 0.83) (Supplementary Picture 5b). Ruzhinji rwemetabolites idzi (5 kubva pa7) i acylcarnitines, iyo inoita basa rakakosha mu fatty acid oxidation pathways.
Sezvakaratidzwa mukushanda kwemabasa kunoratidzwa muMufananidzo 2 a, ma biomarker ekupatsanura ma nodule akawanikwa achishandisa ma least absolute shrinkage operators uye sarudzo zvichibva pa 33 differential metabolites akaonekwa muLA (n = 136) uye BN (n = 170). Best combination of variables (LASSO) - binary logistic regression model. Ten-fold cross-validation yakashandiswa kuyedza kuvimbika kwemuenzaniso. Variable selection uye parameter regularization zvinogadziriswa ne likelihood maximization penalty ne parameter λ24. Global metabolomics analysis yakaitwawo yoga mu internal validation (n = 104) uye external validation (n = 111) groups kuti vaedze classification performance ye discriminant model. Nekuda kweizvozvo, 27 metabolites mu discovery set dzakaonekwa se best discriminant model ine largest mean AUC value (Mufananidzo 2b), pakati padzo 9 dzakawedzera activity uye 18 activity yakaderera muLA zvichienzaniswa neBN (Mufananidzo 2c).
Mafambiro ekushanda ekuvaka pulmonary nodule classifier, kusanganisira kusarudza panel yakanakisa ye serum metabolites mu discovery set uchishandisa binary logistic regression model kuburikidza ne ten-fold cross-validation uye kuongorora predictive performance muma internal ne external validation sets. b Cross-validation statistics ye LASSO regression model ye metabolic biomarker selection. Nhamba dzakapihwa pamusoro dzinomiririra avhareji yehuwandu hwe biomarkers dzakasarudzwa pa λ yakapihwa. Mutsetse mutsvuku une madotsi unomiririra avhareji yeAUC value pa lambda inoenderana. Gray error bars dzinomiririra minimum uye maximum AUC values. Mutsetse une madotsi unoratidza modhi yakanakisa ine 27 biomarkers dzakasarudzwa. AUC, nzvimbo iri pasi pe receiver operating characteristic (ROC) curve. c Peta changes ye 27 selected metabolites mu LA group zvichienzaniswa ne BN group mu discovery group. Red column - activation. Blue column i decrease. d–f Receiver operating characteristic (ROC) curves inoratidza simba re discriminant model zvichibva pa 27 metabolite combinations muma discovery, internal, uye external validation sets. Source data inopiwa muchimiro chema source data files.
Modhi yekufanotaura yakagadzirwa zvichibva paweighted regression coefficients yeaya 27 metabolites (Supplementary Table 3). Kuongororwa kweROC kwakavakirwa paaya 27 metabolites kwakaburitsa nzvimbo iri pasi pecurve (AUC) kukosha kwe0.933, discovery group sensitivity yaive 0.868, uye specificity yaive 0.859 (Mufananidzo 2d). Zvichakadaro, pakati pe38 annoted differential metabolites pakati peLA neHC, seti yemametabolites gumi nematanhatu yakawana AUC ye0.902 ine sensitivity ye0.801 uye specificity ye0.856 mukusiyanisa LA neHC (Supplementary Picture 6a-c). AUC values yakavakirwa pamiganho yakasiyana yekuchinja kwefold ye differential metabolites yakaenzaniswawo. Takaona kuti modhi yekupatsanura yakashanda zvakanaka mukusiyanisa LA neBN (HC) apo fold change level yakaiswa ku1.2 ichienzaniswa ne1.5 kana 2.0 (Supplementary Picture 7a,b). Muenzaniso wekupatsanura, wakavakirwa pamapoka makumi maviri nemanomwe emetabolite, wakasimbiswazve mumakoho emukati nekunze. AUC yaive 0.915 (sensitivity 0.867, specificity 0.811) yekusimbisa mukati uye 0.945 (sensitivity 0.810, specificity 0.979) yekusimbisa kunze (Mufananidzo 2e, f). Kuti vaongorore kushanda zvakanaka kwerabhoritari, sampuli makumi mana kubva kuboka rekunze dzakaongororwa murabhoritari yekunze sezvakatsanangurwa muchikamu cheMethods. Kurongeka kwekupatsanura kwakawana AUC ye0.925 (Mufananidzo Wekuwedzera 8). Nekuti lung squamous cell carcinoma (LUSC) ndiyo yechipiri inonyanya kuzivikanwa yekenza yemapapu isiri diki (NSCLC) mushure me lung adenocarcinoma (LUAD), takaedzawo kushanda kwakasimbiswa kwemaprofiles emetabolic. BN uye nyaya gumi nematanhatu dzeLUSC. AUC yekusarurana pakati peLUSC neBN yaive 0.776 (Mufananidzo Wekuwedzera 9), zvichiratidza kugona kwakaderera zvichienzaniswa nekusarurana pakati peLUAD neBN.
Zvidzidzo zvakaratidza kuti saizi yemapundu pamifananidzo yeCT ine hukama hwakanaka nemukana wekuva negomarara uye ichiri chinhu chikuru chinoratidza kurapwa kwemapundu25,26,27. Kuongororwa kwedata kubva kuboka guru reongororo yeNELSON kwakaratidza kuti njodzi yekuva negomarara muvanhu vane mapundu <5 mm yakanga yakafanana neyevanhu vasina mapundu28. Nokudaro, saizi shoma inoda kuongororwa kweCT nguva dzose i5 mm, sezvakakurudzirwa neBritish Thoracic Society (BTS), uye 6 mm, sezvakakurudzirwa neFleischner Society 29. Zvisinei, mapundu akakura kupfuura 6 mm uye asina zviratidzo zvakajeka, anonzi indeterminate pulmonary nodules (IPN), achiri dambudziko guru mukuongorora nekutarisira mukiriniki30,31. Takazoongorora kana saizi yemapundu yakakanganisa masiginecha emetabolomic tichishandisa samples dzakaunganidzwa kubva kuboka rekutsvaga nekusimbisa mukati. Tichitarisa pane 27 validated biomarkers, takatanga taenzanisa maPCA profiles eHC neBN sub-6 mm metabolomes. Takaona kuti mapoinzi mazhinji edata reHC neBN aipindirana, zvichiratidza kuti mazinga emetabolite muropa aive akafanana mumapoka ese ari maviri (Mufananidzo 3a). Mamepu ezviratidzo muhukuru hwakasiyana akaramba akachengetedzwa muBN neLA (Mufananidzo 3b, c), nepo kupatsanurwa kwakaonekwa pakati pemapundu ane njodzi neasina njodzi muhukuru hwe6-20 mm (Mufananidzo 3d). Boka iri raive neAUC ye0.927, specificity ye0.868, uye sensitivity ye0.820 yekufanotaura kenza yemapundu ine hurefu hwe6 kusvika 20 mm (Mufananidzo 3e, f). Zvatakawana zvinoratidza kuti mugadziri anogona kubata shanduko dzemetabolism dzinokonzerwa nekuchinja kwekutanga kwekenza, zvisinei nehukuru hwemapundu.
Kuenzanisa kwePCA profiles pakati pemapoka akasarudzwa zvichibva pane metabolic classifier ye27 metabolites. CC neBN < 6 mm. b BN < 6 mm vs BN 6–20 mm. muLA 6–20 mm zvichienzaniswa 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 characteristic (ROC) curve inoratidza discriminant model performance yema nodules 6–20 mm. f Probability values yakaverengerwa zvichibva pane logistic regression model yema nodules anoyera 6–20 mm. Mutsetse une grey dotted unomiririra optimal cutoff value (0.455). Nhamba dziri pamusoro dzinomirira chikamu chezviitiko zvinofungidzirwa kuLos Angeles. Shandisa bvunzo yeT yeMudzidzi ine miswe miviri. PCA, ongororo yezvikamu zvikuru. Nzvimbo yeAUC iri pasi pekongiri. Data rekwakabva rinopihwa muchimiro chemafaira edata rekwakabva.
Mienzaniso mina (yemakore 44-61) ine saizi dzakafanana dzemapundu emapapu (7-9 mm) yakasarudzwa zvakare kuratidza mashandiro emuenzaniso wekufanotaura kwekenza (Mufananidzo 4a, b). Pakuongororwa kwekutanga, Nyaya 1 yakaratidza sebundu rakasimba rine calcification, chinhu chine chekuita nekushaya simba, nepo Nyaya 2 yakaratidza sebundu rakasimba zvishoma risina zviratidzo zvakajeka. Kuongororwa katatu kweCT kwakatevera kwakaratidza kuti nyaya idzi dzakaramba dzakagadzikana kwemakore mana uye nekudaro dzakaonekwa sebundu risina simba (Mufananidzo 4a). Zvichienzaniswa nekuongorora kwekiriniki kweserial CT scans, kuongororwa kwese kweserum metabolite ne current classifier model nekukurumidza uye nemazvo kwakaona mabundu aya asina simba zvichibva pazvirambidzo zvekufungidzira (Tafura 1). Mufananidzo 4b muchiitiko che3 unoratidza bundu rine zviratidzo zvekudzoreredzwa kwepleural, izvo zvinowanzobatanidzwa nekenza32. Nyaya 4 inoratidza sebundu rakasimba zvishoma risina humbowo hwechikonzero chisina kunaka. Nyaya idzi dzese dzakafanotaurwa sebundu rine simba zvichienderana nemuenzaniso weclassifier (Tafura 1). Kuongororwa kwekenza yemapapu kwakaratidzwa nekuongorora histopathological mushure mekuvhiyiwa kwekubvisa mapapu (Mufananidzo 4b). Pakuongorora kwekunze, mugadziri wemetabolism akafanotaura nemazvo zviitiko zviviri zvemapundu emapapu asina kujeka akakura kupfuura 6 mm (Mufananidzo Wekuwedzera 10).
Mifananidzo yeCT yehwindo remapapu remaaxial ezvirwere zviviri zvemapundu asina njodzi. Muchiitiko chekutanga, CT scan mushure memakore mana yakaratidza bundu rakasimba rakagadzikana rinosvika 7 mm rine calcification muchikamu chezasi chekurudyi. Muchiitiko chechipiri, CT scan mushure memakore mashanu yakaratidza bundu rakasimba, rakati sandara rine dhayamita ye7 mm muchikamu chezasi chekurudyi. b Mifananidzo yeCT yeAxial window uye zvidzidzo zvinoenderana zvezvirwere zvezviitiko zviviri zve stage I adenocarcinoma isati yabviswa mapapu. Nyaya yechitatu yakaratidza bundu rine dhayamita ye8 mm muchikamu chezasi chekurudyi chine pleural retraction. Nyaya yechina yakaratidza bundu regirazi repasi rakasimba rinosvika 9 mm muchikamu chezasi chekuruboshwe. Kutsveyama kweHematoxylin neeosin (H&E) kwetishu yemapapu yakabviswa (scale bar = 50 μm) kuratidza maitiro ekukura kweacinar yemapapu adenocarcinoma. Miseve inoratidza mapundu akaonekwa pamifananidzo yeCT. Mifananidzo yeH&E inomiririra minda yakawanda (>3) microscopic yakaongororwa nenyanzvi yezvirwere.
Kana zvabatanidzwa, zvatakawana zvinoratidza kukosha kwezvibodzwa zve serum metabolite mukuongorora kwakasiyana kwemapundu emapapu, izvo zvinogona kukonzera matambudziko pakuongorora CT screening.
Zvichibva pane imwe panel yemetabolite yakasimbiswa, takatsvaga kuona hukama hwezvinhu zvipenyu hwekuchinja kukuru kwemetabolism. Kuongororwa kweKEGG pathway enrichment naMetaboAnalyst kwakaratidza nzira 6 dzakajairika dzakachinjwa zvikuru pakati pemapoka maviri akapihwa (LA vs. HC naLA vs. BN, yakagadziriswa p ≤ 0.001, effect > 0.01). Shanduko idzi dzakaratidzwa nekukanganisika mu pyruvate metabolism, tryptophan metabolism, niacin uye nicotinamide metabolism, glycolysis, TCA cycle, uye purine metabolism (Mufananidzo 5a). Takabva taita zvakare metabolomics dzakatarwa kuti tione shanduko huru tichishandisa absolute quantification. Kuongororwa kwemetabolites dzakajairika munzira dzakajairwa dzakajairwa ne triple quadrupole mass spectrometry (QQQ) tichishandisa zviyero zvemetabolite zvechokwadi. Hunhu hwehuwandu hwevanhu hwemuenzaniso wekudzidza metabolomics hunowanikwa muSupplementary Table 4. Zvichienderana nemigumisiro yedu yepasi rose metabolomics, ongororo yehuwandu yakaratidza kuti hypoxanthine ne xanthine, pyruvate, uye lactate zvakawedzera mu LA zvichienzaniswa neBN neHC (Mufananidzo 5b, c, p <0.05). Zvisinei, hapana musiyano wakakosha mu metabolites idzi wakawanikwa pakati peBN neHC.
Kuongororwa kweKEGG pathway enrichment ye metabolites dzakasiyana zvakanyanya muboka reLA zvichienzaniswa nemapoka eBN neHC. Globaltest ine tailed two-tailed yakashandiswa, uye p values dzakagadziriswa uchishandisa nzira yeHolm-Bonferroni (yakagadziriswa p ≤ 0.001 uye saizi yemhedzisiro > 0.01). b–d Violin plots inoratidza mazinga ehypoxanthine, xanthine, lactate, pyruvate, uye tryptophan mu serum HC, BN, uye LA zvakatemwa neLC-MS/MS (n = 70 paboka). Mitsetse michena nemitema inoratidza median nequartile, zvichiteerana. e Violin plot inoratidza normalized Log2TPM (transcripts per million) mRNA expression yeSLC7A5 neQPRT mu lung adenocarcinoma (n = 513) zvichienzaniswa ne normal lung tissue (n = 59) muLUAD-TCGA dataset. Bhokisi jena rinomiririra nzvimbo iri pakati pemakota, mutsetse mutema wakatambanuka uri pakati unomiririra pakati, uye mutsetse mutema wakamira unobva mubhokisi unomiririra 95% confidence interval (CI). f Pearson correlation plot yeSLC7A5 neGAPDH expression mu lung adenocarcinoma (n = 513) uye normal lung tissue (n = 59) muTCGA dataset. Nzvimbo yegrey inomiririra 95% CI. r, Pearson correlation coefficient. g Normalized cellular tryptophan levels muA549 cells transfected with nonspecific shRNA control (NC) uye shSLC7A5 (Sh1, Sh2) yakatarwa neLC-MS/MS. Kuongororwa kwestatistike kwesamples shanu dzakazvimiririra mu biologically muboka rega rega kunoratidzwa. h Masero eNADt (total NAD, kusanganisira NAD+ neNADH) muA549 cells (NC) uye SLC7A5 knockdown A549 cells (Sh1, Sh2). Kuongororwa kwestatistike kwesamples nhatu dzakazvimiririra mu biologically muboka rega rega kunoratidzwa. Basa reGlycolytic remasero eA549 risati rasvika uye mushure mekudonha kweSLC7A5 rakayerwa ne extracellular acidification rate (ECAR) (n = 4 biologically independent samples per group). 2-DG,2-deoxy-D-glucose. Two-tailed Student's t test yakashandiswa mu (b–h). Mu (g–i), error bars dzinomiririra avhareji ± SD, bvunzo yega yega yakaitwa katatu yakazvimiririra uye mhedzisiro yacho yakafanana. Source data inopihwa muchimiro chemafaira esource data.
Tichifunga nezvemhedzisiro yakakosha yekuchinja kwemetabolism yetryptophan muboka reLA, takaongororawo huwandu hwetryptophan muropa mumapoka eHC, BN, uye LA tichishandisa QQQ. Takaona kuti serum tryptophan yakaderedzwa muLA zvichienzaniswa neHC kana BN (p < 0.001, Mufananidzo 5d), izvo zvinoenderana nezvakawanikwa kare zvekuti huwandu hwetryptophan hunotenderera hwakaderera muvarwere vane kenza yemapapu pane muvanhu vane hutano kubva kuboka rekutonga33,34,35. Imwe ongororo yakashandisa PET/CT tracer 11C-methyl-L-tryptophan yakawana kuti nguva yekuchengetedza signal yetryptophan mumatishu ekenza yemapapu yakawedzera zvakanyanya zvichienzaniswa nezvipembenene zvisina njodzi kana matishu akajairwa36. Tinofungidzira kuti kudzikira kwetryptophan muserum yeLA kunogona kuratidza active tryptophan uptake nemasero ekenza yemapapu.
Zvinozivikanwawo kuti chibereko che kynurenine pathway ye tryptophan catabolism i NAD+37,38, inova substrate yakakosha ye reaction ye glyceraldehyde-3-phosphate ne 1,3-bisphosphoglycerate mu glycolysis39. Kunyange hazvo zvidzidzo zvekare zvakatarisa pabasa re tryptophan catabolism mukudzora immune system, takatsvaga kujekesa kubatana kuripo pakati pe tryptophan dysregulation uye glycolytic pathways yakaonekwa muchidzidzo chazvino. Solute transporter family 7 member 5 (SLC7A5) inozivikanwa se tryptophan transporter43,44,45. Quinolinic acid phosphoribosyltransferase (QPRT) i enzyme iri pasi pe kynurenine pathway inoshandura quinolinic acid kuita NAMN46. Kuongororwa kwedata reLUAD TCGA kwakaratidza kuti SLC7A5 neQPRT zvakawedzera zvakanyanya mu tumor tichienzaniswa ne normal tissue (Mufananidzo 5e). Kuwedzera uku kwakaonekwa muzvikamu zvekutanga ne zvechipiri pamwe nezvikamu zvechitatu ne zvechina zve lung adenocarcinoma (Mufananidzo wekuwedzera 11), zvichiratidza kusagadzikana kwekutanga mu tryptophan metabolism inobatanidzwa ne bundu.
Pamusoro pezvo, data reLUAD-TCGA rakaratidza hukama hwakanaka pakati peSLC7A5 neGAPDH mRNA expression muvarwere vegomarara (r = 0.45, p = 1.55E-26, Mufananidzo 5f). Kusiyana neizvi, hapana hukama hwakakosha hwakawanikwa pakati pemagene signatures akadaro mumatishu emapapu akajairwa (r = 0.25, p = 0.06, Mufananidzo 5f). Kudonhedzwa kweSLC7A5 (Mufananidzo Wekuwedzera 12) mumasero eA549 kwakaderedza zvakanyanya masero etryptophan neNAD(H) (Mufananidzo 5g,h), zvichikonzera attenuated glycolytic activity sezvakayerwa ne extracellular acidification rate (ECAR) (Mufananidzo 1). 5i). Saka, zvichibva pakuchinja kwe metabolic mu serum uye in vitro detection, tinofungidzira kuti tryptophan metabolism inogona kugadzira NAD+ kuburikidza ne kynurenine pathway uye inoita basa rakakosha mukusimudzira glycolysis mugomarara remapapu.
Zvidzidzo zvakaratidza kuti huwandu hwakawanda hwemapundu emumapapu asina kujeka akaonekwa neLDCT hunogona kukonzera kudiwa kwekuongororwa kwakawanda kwakadai sePET-CT, biopsy yemapapu, uye kurapwa kwakanyanya nekuda kwekuongororwa kwekenza yemudumbu.31 Sezvakaratidzwa muMufananidzo 6, chidzidzo chedu chakaratidza boka remametabolites emuropa ane mukana wekuongorora anogona kuvandudza kupatsanurwa kwenjodzi uye kutarisirwa kwemapundu emumapapu akaonekwa neCT.
Mapundu emapapu anoongororwa uchishandisa computed tomography (LDCT) ine mifananidzo inoratidza zvikonzero zvisina njodzi kana zvakaipa. Mhedzisiro isina chokwadi yemapundu inogona kutungamira kushanyirwa kakawanda, kupindira kusingakoshi, uye kurapwa zvakanyanya. Kubatanidzwa kwezvikamu zve metabolic muropa zvine kukosha kwekuongorora kunogona kuvandudza kuongororwa kwenjodzi uye kutarisirwa kwepundu dzemapapu. PET positron emission tomography.
Data kubva kuongororo yeUS NLST pamwe neongororo yeEuropean NELSON inoratidza kuti kuongorora mapoka ane njodzi huru ne computed tomography (LDCT) shoma kunogona kuderedza kufa kwevanhu vane kenza yemapapu1,3. Zvisinei, kuongororwa kwenjodzi uye kurapwa kwekiriniki kwehuwandu hwakawanda hwemapundu emapapu akaonekwa neLDCT kunoramba kuri kwakanyanya kuoma. Chinangwa chikuru ndechekugadzirisa kupatsanurwa kwakakodzera kwemaitiro aripo eLDCT-based protocols nekubatanidza biomarkers dzakavimbika.
Mamwe mamorekuru ebiomarker, akadai semametabolites eropa, akaonekwa nekuenzanisa kenza yemapapu nevanodzora hutano15,17. Muchidzidzo chazvino, takatarisana nekushandiswa kwekuongorora kwe serum metabolomics kuti tisiyanise pakati pemapundu asina njodzi uye akaipa akaonekwa neLDCT. Takaenzanisa global serum metabolome of healthy control (HC), mapundu asina njodzi emapapu (BN), uye samples dze stage I lung adenocarcinoma (LA) tichishandisa ongororo yeUPLC-HRMS. Takaona kuti HC neBN zvaive nema metabolic profiles akafanana, nepo LA yakaratidza shanduko huru zvichienzaniswa neHC neBN. Takaona seti mbiri dzemaseru metabolites anosiyanisa LA kubva kuHC neBN.
Chirongwa cheLDCT chekuziva maronda asina njodzi uye ane kenza chinonyanya kuenderana nehukuru, huwandu, chimiro uye mwero wekukura kwemaronda nekufamba kwenguva. Zvidzidzo zvekare zvakaratidza kuti hukuru hwemaronda hwakabatana zvikuru nemukana wekenza yemapapu. Kunyangwe muvarwere vane njodzi huru, njodzi yekenza mumaronda ari pasi pe6 mm i <1%. Njodzi yekenza yemaronda ari pakati pe6 kusvika 20 mm inotangira pa8% kusvika 64%30. Nokudaro, Fleischner Society inokurudzira dhayamita ye6 mm yekutevera CT nguva dzose. 29 Zvisinei, kuongororwa kwenjodzi uye manejimendi emaronda emapapu asina kujeka (IPN) akakura kupfuura 6 mm hazvina kuitwa zvakakwana 31. Kutarisirwa kwechirwere chemwoyo chekuzvarwa nacho kunowanzo enderana nekumirira wakangwarira nekutarisa CT nguva nenguva.
Zvichibva pa metabolome yakaongororwa, takaratidza kekutanga kuti pane kubatana kwezviratidzo zve metabolomic pakati pevanhu vane hutano nema nodules asina njodzi <6 mm. Kufanana kwezvipenyu kunoenderana nezvakawanikwa kare zveCT kuti njodzi yekenza yema nodules <6 mm yakaderera seyevanhu vasina ma nodules.30 Zvinofanira kucherechedzwa kuti zvatakawana zvinoratidzawo kuti ma nodules asina njodzi <6 mm uye ≥6 mm akafanana zvakanyanya mu metabolomic profiles, zvichiratidza kuti tsananguro inoshanda ye etiology isina njodzi inowirirana pasinei nehukuru hwema nodules. Saka, mapaneru e serum metabolite ekuongorora emazuva ano anogona kupa bvunzo imwe chete seyedzo yekurega kana ma nodules akaonekwa pakutanga paCT uye anogona kuderedza kutarisa kwe serial. Panguva imwe chete, panel imwechete ye metabolic biomarkers yakasiyanisa ma nodules ane njodzi ≥6 mm muhukuru kubva kuma nodules asina njodzi uye yakapa kufanotaura kwakarurama kwema IPNs ane saizi yakafanana uye morphological features isina kujeka pamifananidzo yeCT. Iyi serum metabolism classifier yakaita zvakanaka mukufanotaura kenza yema nodules ≥6 mm neAUC ye0.927. Kana zvabatanidzwa, zvatakawana zvinoratidza kuti zviratidzo zvakasiyana zve metabolomic muropa zvinogona kuratidza shanduko dzekutanga dzema metabolic dzinokonzereswa negomarara uye zvinogona kuva nemhedzisiro yengozi, zvisinei nehukuru hwemapundu.
Zvinonyanya kukosha, lung adenocarcinoma (LUAD) uye squamous cell carcinoma (LUSC) ndidzo mhando huru dzekenza yemapapu isiri diki (NSCLC). Tichifunga kuti LUSC ine hukama hwakasimba nekushandiswa kwefodya47 uye LUAD ndiyo inonyanya kuzivikanwa yemapundu emapapu akaonekwa paCT screening48, modhi yedu yeclassifier yakagadzirirwa samples dze stage I adenocarcinoma. Wang nevamwe vake vakatarisawo paLUAD uye vakaona ma lipid signature mapfumbamwe vachishandisa lipidomics kusiyanisa kenza yemapapu yepakutanga kubva kune vanhu vane hutano17. Takaedza modhi yeclassifier iripo pazviitiko gumi nematanhatu zve stage I LUSC uye mapundu makumi manomwe nemana asina njodzi uye takaona kururama kwekufanotaura kweLUSC kwakaderera (AUC 0.776), zvichiratidza kuti LUAD neLUSC zvinogona kunge zvine ma metabolomic signatures azvo. Zvechokwadi, LUAD neLUSC zvakaratidzwa kuti zvakasiyana mu etiology, biological origin uye genetic aberrations49. Saka, mamwe marudzi ehistology anofanira kuiswa mumhando dzekudzidzisa kuona kenza yemapapu zvichibva pahuwandu hwevanhu muzvirongwa zvekuongorora.
Pano, takaona nzira nhanhatu dzinonyanya kuchinjwa mu lung adenocarcinoma kana tichienzanisa nevanodzora hutano uye ma nodules asina njodzi. Xanthine ne hypoxanthine ndiwo ma metabolites akajairika e purine metabolic pathway. Zvichienderana nezvatakawana, ma intermediates ane chekuita ne purine metabolism akawedzera zvakanyanya mu serum kana mumatishu evarwere vane lung adenocarcinoma kana tichienzanisa nevanodzora hutano kana varwere vari padanho rekutanga rekupinda muropa15,50. Mazinga e serum xanthine ne hypoxanthine akakwira anogona kuratidza anabolism inodiwa nemasero ekenza ari kuwedzera nekukurumidza. Kusashanda zvakanaka kwe glucose metabolism chiratidzo chinozivikanwa che cancer metabolism51. Pano, takaona kuwedzera kukuru kwe pyruvate ne lactate mu LA group zvichienzaniswa ne HC ne BN group, izvo zvinoenderana nemishumo yapfuura ye glycolytic pathway abnormalities mu serum metabolome profiles yevarwere vasiri small cell lung cancer (NSCLC) uye vanodzora hutano. mhedzisiro yacho yakafanana52,53.
Zvakakosha, takaona hukama hwakasiyana pakati pe pyruvate ne tryptophan metabolism mu serum ye lung adenocarcinomas. Mazinga e serum tryptophan akadzikira mu LA group zvichienzaniswa ne HC kana BN group. Zvinofadza kuti, ongororo yakakura yapfuura yakashandisa prospective cohort yakawana kuti mazinga akaderera e circulating tryptophan aibatanidzwa nekuwedzera kwenjodzi yekenza yemapapu 54. Tryptophan i amino acid inokosha yatinowana kubva muchikafu. Tinogumisa kuti low tryptophan mu serum mu lung adenocarcinoma inogona kuratidza kukurumidza kupera kwe metabolite iyi. Zvinozivikanwa kuti mhedzisiro yekupedzisira ye tryptophan catabolism kuburikidza ne kynurenine pathway ndiyo sosi ye de novo NAD+ synthesis. Nekuti NAD+ inogadzirwa zvakanyanya kuburikidza ne salvage pathway, kukosha kweNAD+ mu tryptophan metabolism muhutano nezvirwere kuchiri kufanira kuzivikanwa46. Kuongorora kwedu dhatabhesi reTCGA kwakaratidza kuti kuratidzwa kwetryptophan transporter solute transporter 7A5 (SLC7A5) kwakawedzera zvakanyanya mu lung adenocarcinoma zvichienzaniswa nemaitiro akajairwa uye kwakabatana zvakanaka nekuratidzwa kweglycolytic enzyme GAPDH. Zvidzidzo zvekare zvakatarisa zvakanyanya pabasa retryptophan catabolism mukudzvinyirira antitumor immune response40,41,42. Pano tinoratidza kuti kudziviswa kwe tryptophan uptake nekudonhedza SLC7A5 mumasero ekenza yemapapu kunoguma nekuderera kwemazinga emasero eNAD uye kudzikira kwe glycolytic activity panguva imwe chete. Mukupfupisa, chidzidzo chedu chinopa hwaro hwebiological hwekuchinja kwe serum metabolism kwakabatana nekuchinja kwe lung adenocarcinoma.
Kuchinja kweEGFR ndiko kunonyanya kukonzera kuchinja kwemasero muvarwere vane NSCLC. Mukudzidza kwedu, takaona kuti varwere vane kuchinja kweEGFR (n = 41) vaiva nemaprofiles e metabolomic akafanana nevarwere vane wild-type EGFR (n = 31), kunyange zvazvo takawana kudzikira kwemazinga emuropa evamwe varwere vane EGFR mutant muvarwere vane acylcarnitine. Basa rakasimbiswa re acylcarnitines nderekutakura mapoka e acyl kubva ku cytoplasm kuenda ku mitochondrial matrix, zvichikonzera oxidation ye fatty acids kuti igadzire simba 55. Zvichienderana nezvatakawana, chidzidzo chechangobva kuitwa chakaratidzawo maprofiles akafanana e metabolome pakati peEGFR mutant neEGFR wild-type tumors nekuongorora metabolome yepasi rose ye102 lung adenocarcinoma tissue samples50. Zvinofadza kuti, huwandu hwe acylcarnitine hwakawanikwawo muboka re EGFR mutant. Nokudaro, kana shanduko mumazinga e acylcarnitine dzichiratidza shanduko dze metabolic dzakakonzereswa neEGFR uye nzira dziri pasi pemamorekuru dzinogona kukodzera kudzidza kwakawanda.
Mukupedzisa, chidzidzo chedu chinogadza nzira yekuongorora metabolic muropa yekuongorora maronda emapapu uye chinokurudzira nzira yekushanda inogona kuvandudza kuongororwa kwenjodzi uye kuita kuti kurapwa kuve nyore zvichibva pakuongorora kweCT scan.
Chidzidzo ichi chakatenderwa neKomiti yeMaitiro eSun Yat-sen University Cancer Hospital, Chipatara Chekutanga Chinobatanidzwa cheSun Yat-sen University, uye Komiti yeMaitiro eZhengzhou University Cancer Hospital. Mumapoka ekutsvaga nekusimbisa mukati, masera 174 kubva kuvanhu vane hutano uye masera 244 kubva kumanodules asina njodzi akatorwa kubva kuvanhu vaiongororwa gore rega rega paDhipatimendi reCancer Control and Prevention, Sun Yat-sen University Cancer Center, uye masera 166 asina njodzi. Serum. Manodules emapapu eStage I akatorwa kubva kuSun Yat-sen University Cancer Center. Muboka rekusimbisa kunze, masera 48 emanodules asina njodzi, masera 39 emapapu echikamu chekutanga kubva kuChipatara Chekutanga cheSun Yat-sen University, uye masera 24 emapapu echikamu chekutanga kubva kuChipatara Chekutanga cheZhengzhou Cancer. Sun Yat-sen University Cancer Center yakaunganidzawo nyaya gumi nematanhatu dzekenza yemapapu yechikamu chekutanga (stage I squamous cell lung cancer) kuti iongorore kugona kwekuongorora kwe metabolic classifier (hunhu hwemurwere hunoratidzwa muSupplementary Table 5). Samples kubva kuboka rekutsvaga neboka re internal validation dzakatorwa pakati paNdira 2018 naChivabvu 2020. Samples dzeboka re external validation dzakatorwa pakati paNyamavhuvhu 2021 naGumiguru 2022. Kuti kuderedze rusaruro rwevanhurume nevanhukadzi, nhamba dzakaenzana dzenyaya dzevarume nevakadzi dzakapihwa boka rega rega. Discovery Team neInternal Review Team. Vanhurume nevanhukadzi vakatorwa vakaongororwa zvichibva pamushumo wavo. Mvumo yakatorwa kubva kune vese vakatora chikamu uye hapana muripo wakapihwa. Vanhu vane benign nodules vaive avo vane CT scan score yakagadzikana pamakore maviri kusvika mashanu panguva yekuongorora, kunze kwenyaya imwe chete kubva kumuenzaniso we external validation, iyo yakatorwa isati yavhiyiwa uye yakaonekwa ne histopathology. Kunze kwe chronic bronchitis. Nyaya dze lung adenocarcinoma dzakatorwa mapapu asati abviswa uye dzakasimbiswa ne pathological diagnosis. Masampuli eropa rekutsanya akaunganidzwa mumachubhu ekuparadzanisa serum pasina mishonga inorwisa ropa. Masampuli eropa akagwamba kweawa imwe chete patembiricha yemukamuri uye akazoiswa mu centrifuge pa 2851 × g kwemaminitsi gumi pa 4°C kuti atore serum supernatant. Ma serum aliquots akaomeswa nechando pa -80°C kusvika abviswa metabolite. Dhipatimendi reKudzivirira Cancer uye Kuongororwa Kwezvokurapa reSun Yat-sen University Cancer Center rakaunganidza serum kubva kuvapi 100 vane hutano hwakanaka, kusanganisira nhamba yakaenzana yevarume nevakadzi vane makore 40 kusvika 55. Mavhoriyamu akaenzana emuenzaniso wega wega wevapi akasanganiswa, serum yakabuda yakatorwa uye yakachengetwa pa -80°C. Musanganiswa we serum wakashandiswa senzira yekutarisa kunaka uye kuyera data.
Seramu yereferensi uye sampuli dzebvunzo dzakanyungudutswa uye metabolites dzakabviswa uchishandisa nzira yekubvisa yakabatana (MTBE/methanol/mvura) 56. Muchidimbu, 50 μl yeseramu yakasanganiswa ne225 μl yemethanol inotonhora ne750 μl yemethyl tert-butyl ether inotonhora nechando (MTBE). Sanganisa musanganiswa uye isa muchando kweawa imwe chete. Samples dzakazosanganiswa uye vortex yakasanganiswa ne188 μl yemvura yeMS-grade ine zviyero zvemukati (13C-lactate, 13C3-pyruvate, 13C-methionine, uye 13C6-isoleucine, yakatengwa kubva kuCambridge Isotope Laboratories). Musanganiswa wacho wakabva waiswa centrifuge pa15,000 × g kwemaminitsi gumi pa4 °C, uye chikamu chepazasi chakachinjirwa mumachubhu maviri (125 μL imwe neimwe) kuti pave neLC-MS analysis mumhando dzakanaka nedzisina kunaka. Pakupedzisira, sampuro yacho yakapwanyika kusvika yaoma muchina unopinza mhepo unokurumidza.
Mametabolites akaomeswa akagadziriswazve mu 120 μl ye 80% acetonitrile, akasvinwa kwemaminitsi mashanu, uye akasvinwa pa 15,000 × g kwemaminitsi gumi pa 4°C. Supernatants dzakaiswa mumabhodhoro egirazi e amber ane microinserts yezvidzidzo zve metabolomics. Untargeted metabolomics analysis pa ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) platform. Metabolites dzakaparadzaniswa uchishandisa Dionex Ultimate 3000 UPLC system uye ACQUITY BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters). Mu positive ion mode, ma mobile phases aive 95% (A) uye 50% acetonitrile (B), imwe neimwe ine 10 mmol/L ammonium acetate uye 0.1% formic acid. Mumamiriro asina kunaka, zvikamu zve mobile A neB zvaive ne 95% uye 50% acetonitrile, zvichiteerana, zvikamu zvese zvaive ne 10 mmol/L ammonium acetate, pH = 9. Chirongwa che gradient chaive seizvi: 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. Koramu yakachengetedzwa pa 40°C uye sample pa 10°C mu autosampler. Mwero wekuyerera waive 0.3 ml/min, huwandu hwe injection hwaive 3 μl. Chiyero cheQ-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) chine electrospray ionization (ESI) source chakashandiswa mu full scan mode uye chakabatana ne ddMS2 monitoring mode kuti chiunganidze data rakawanda. MS parameters dzakaiswa seizvi: spray voltage +3.8 kV/- 3.2 kV, capillary temperature 320°C, shielding gas 40 arb, assistant gas 10 arb, probe heater temperature 350°C, scanning range 70–1050 m/h, resolution. 70 000. Data rakatorwa uchishandisa Xcalibur 4.1 (Thermo Fisher Scientific).
Kuti vaongorore mhando yedata, sampuli dzakaunganidzwa dzekudzora mhando (QC) dzakagadzirwa nekubvisa 10 μL aliquots ye supernatant kubva musampuli yega yega. Majekiseni matanhatu ekudzora mhando akaongororwa pakutanga kwe analytical sequence kuti vaongorore kugadzikana kweUPLC-MS system. Samples dzekudzora mhando dzinozoiswa nguva nenguva mu batch. Ma batch ese gumi nerimwe ema serum samples muchidzidzo ichi akaongororwa neLC-MS. Zvimwe zvikamu zvemusanganiswa we serum pool kubva kune 100 healthy donors zvakashandiswa se reference material muma batches akasiyana kuti vatarise maitiro ekubvisa uye kugadzirisa mhedzisiro ye batch-to-batch. Kuongororwa kwe metabolomics isina kunangwa ye discount cohort, internal validation cohort, uye external validation cohort kwakaitwa paMetabolomics Center yeSun Yat-sen University. Laboratory yekunze yeGuangdong University of Technology Analysis and Testing Center yakaongororawo sampuli makumi mana kubva ku external cohort kuti vaedze mashandiro e classifier model.
Mushure mekubviswa nekugadziriswazve, huwandu hwakazara hwemametabolites emuropa hwakayerwa uchishandisa ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) ne electrospray ionization (ESI) source mu multiple reaction monitoring (MRM) mode. ACQUITY BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters) yakashandiswa kuparadzanisa metabolites. Mobile phase yaive ne 90% (A) ne 5% acetonitrile (B) ine 10 mmol/L ammonium acetate uye 0.1% ammonia solution. Gradient program yaive seinotevera: 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. Koramu yacho yakachengetedzwa pa40 °C uye sample pa10 °C mu autosampler. Mwero wekuyerera waive 0.3 mL/min uye huwandu hwejekiseni hwaive 1 μL. MS parameters dzakaiswa seizvi: capillary voltage ±3.5 kV, nebulizer pressure 35 psi, sheath gas flow 12 L/min, sheath gas temperature 350°C, drying gas temperature 250°C, uye drying gas flow 14 l/min. Kuchinja kweMRM kwetryptophan, pyruvate, lactate, hypoxanthine uye xanthine kwaive 205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 uye 151.0–107. 9 zvichiteerana. Data rakaunganidzwa uchishandisa Mass Hunter B.07.00 (Agilent Technologies). Pamasampuli emuropa, tryptophan, pyruvate, lactate, hypoxanthine, uye xanthine zvakayerwa uchishandisa ma calibration curves e standard mixed solutions. Pamasampuli emasero, huwandu hwe tryptophan hwakagadziriswa kusvika pachiyero chemukati uye huwandu hweprotein yemasero.
Kubviswa kwenhamba yepamusoro (m/z nenguva yekuchengeta (RT)) kwakaitwa uchishandisa Compound Discovery 3.1 neTraceFinder 4.0 (Thermo Fisher Scientific). Kuti kubviswe mutsauko uripo pakati pemabatch, nhamba yega yega yemuenzaniso wekuyedza yakakamurwa nenhamba yepamusoro yezvinyorwa zvekutarisa kubva mubatch imwe chete kuti iwane huwandu hwakaenzana. Kutsauka kwenhamba yepamusoro kwemazinga emukati asati asvika uye mushure mekugadziriswa kunoratidzwa muSupplementary Table 6. Musiyano uripo pakati pemapoka maviri wakaratidzwa ne false discovery rate (FDR<0.05, Wilcoxon signed rank test) uye fold change (>1.2 kana <0.83). Raw MS data yezvikamu zvakabviswa uye data re serum-corrected MS rinoratidzwa muSupplementary Data 1 neSupplementary Data 2, zvichiteerana. Peak annotation yakaitwa zvichibva pamatanho mana akatsanangurwa ekuzivikanwa, kusanganisira metabolites akawanikwa, compounds dzinofungidzirwa, compounds dzinofungidzirwa, uye compounds dzisingazivikanwi 22. Zvichibva pakutsvaga kwedatabase muCompound Discovery 3.1 (mzCloud, HMDB, Chemspider), makemikari ezvipenyu ane MS/MS matching validated standards kana exact match annotations mu mzCloud (score > 85) kana Chemspider akazosarudzwa se intermediates pakati pe differential metabolome. Peak annotations yechinhu chimwe nechimwe inosanganisirwa muSupplementary Data 3. MetaboAnalyst 5.0 yakashandiswa pakuongorora univariate ye sum-normalized metabolite abundance. MetaboAnalyst 5.0 yakaongororawo KEGG pathway enrichment analysis zvichibva pane metabolites dzakasiyana. Principal component analysis (PCA) uye partial least squares discriminant analysis (PLS-DA) zvakaongororwa uchishandisa ropls software package (v.1.26.4) ne stack normalization uye autoscaling. Iyo optimal metabolite biomarker model yekufanotaura nodule mondancy yakagadzirwa uchishandisa binary logistic regression ne least absolute shrinkage and selection operator (LASSO, R package v.4.1-3). Kushanda kwemuenzaniso wekusarudza muzvikamu zvekuongorora nekusimbisa kwakatsanangurwa nekufungidzira AUC zvichibva pakuongorora kweROC zvichienderana nepakeji yepROC (v.1.18.0.). Muganho wepamusoro wemukana wakawanikwa zvichibva pane yakanyanya Youden index yemuenzaniso (sensitivity + specificity - 1). Samples dzine kukosha kuri pasi kana kukuru kupfuura threshold dzichafanotaurwa se benign nodules uye lung adenocarcinoma, zvichiteerana.
Masero eA549 (#CCL-185, American Type Culture Collection) akakurira muF-12K medium ine 10% FBS. Ma "short hairpin RNA" (shRNA) sequences akananga kuSLC7A5 uye nontargeting control (NC) akaiswa mu lentiviral vector pLKO.1-puro. Ma "antisense sequences" e shSLC7A5 ndeaya anotevera: Sh1 (5′-GGAGAAACCTGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGGAACTAT-3′). Ma "antibodies" eSLC7A5 (#5347) uye tubulin (#2148) akatengwa kubva kuCell Signaling Technology. Ma "antibodies" eSLC7A5 netubulin akashandiswa pakusanganiswa kwe1:1000 kweWestern blot analysis.
Kuongororwa kweSeahorse XF Glycolytic Stress Test kunoyera mazinga e extracellular acidification (ECAR). Mukuongorora, glucose, oligomycin A, uye 2-DG zvakapihwa zvakatevedzana kuti zviongorore kugona kwemasero eglycolytic sezvakayerwa neECAR.
Masero eA549 akapinzwa ne non-targeting control (NC) uye shSLC7A5 (Sh1, Sh2) akaputirwa husiku hwese mundiro dzine dhayamita ye10 cm. Masero emetabolites akabviswa ne 1 ml ye 80% methanol ine mvura inotonhora zvikuru. Masero ari mumushonga wemethanol akakweshwa, akaunganidzwa muchubhu itsva, ndokuiswa mu centrifuge pa 15,000 × g kwemaminitsi gumi nemashanu pa 4°C. Kuunganidza 800 µl ye supernatant woomesa uchishandisa high-speed vacuum concentrator. Ma pellets emetabolite akaomeswa akazoongororwa kuti aone tryptophan levels achishandisa LC-MS/MS sezvakatsanangurwa pamusoro apa. Masero eNAD(H) mumasero eA549 (NC ne shSLC7A5) akayerwa achishandisa quantitative NAD+/NADH colorimetric kit (#K337, BioVision) zvichienderana nemirairo yemugadziri. Mapuroteni levels akayerwa pamuenzaniso wega wega kuti huwandu hwemetabolites huenderane nemaitiro akajairika.
Hapana nzira dzezviverengero dzakashandiswa kutanga kuona saizi yemuenzaniso. Zvidzidzo zvekare zvemetabolomics zvainangana nekuwanikwa kwebiomarker15,18 zvakaonekwa sezviyero zvekuona saizi, uye zvichienzaniswa nemishumo iyi, muenzaniso wedu wakanga wakakwana. Hapana mienzaniso yakabviswa muboka rekudzidza. Sampuli dzeserum dzakapihwa zvisina kurongeka kuboka rekutsvaga (zviitiko 306, 74.6%) uye boka rekusimbisa mukati (zviitiko 104, 25.4%) kune zvidzidzo zvemetabolomics zvisina kunangwa. Isu takasarudzawo zvisina kurongeka zviitiko 70 kubva kuboka rega rega kubva muchikamu chekutsvaga chezvidzidzo zvemetabolomics zvakanangwa. Vaongorori vakapofumadzwa kuti vasaone kugoverwa kweboka panguva yekuunganidza nekuongorora data reLC-MS. Kuongororwa kwezviverengero zvedata remetabolomics uye kuyedzwa kwemasero kunotsanangurwa muzvikamu zveResults, Figure Legends, uye Methods. Kuyera kwemasero etryptophan, NADT, uye glycolytic kwakaitwa katatu zvakazvimirira nemigumisiro yakafanana.
Kuti uwane rumwe ruzivo nezve dhizaini yekudzidza, ona Natural Portfolio Report Abstract yakabatana nechinyorwa chino.
Data reMS raw rezvinhu zvakabviswa pamwe nedata reMS rakajairika re serum reference rinoratidzwa muSupplementary Data 1 neSupplementary Data 2, zvichiteerana. Peak annotations ye differential features inoratidzwa muSupplementary Data 3. LUAD TCGA dataset inogona kudhawunirodhwa kubva ku https://portal.gdc.cancer.gov/. Data rekuisa girafu rinowanikwa mudata resource. Data resource rinowanikwa muchinyorwa chino.
Boka reNational Lung Screening Study Group, nezvimwewo. Kuderedza kufa kwevanhu vane kenza yemapapu nekushandisa computed tomography shoma. Northern England. J. Med. 365, 395–409 (2011).
Kramer, BS, Berg, KD, Aberle, DR naProphet, PC Kuongororwa kenza yemapapu uchishandisa helical CT ine dose shoma: mhedzisiro kubva kuNational Lung Screening Study (NLST). J. Med. Screen 18, 109–111 (2011).
De Koning, HJ, nevamwe. Kuderedza kufa kwevanhu vane kenza yemapapu nekuongorora CT yakawanda muongororo yakasarudzika. Northern England. J. Med. 382, 503–513 (2020).
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