Nchọpụta dị iche iche nke nodules pulmonary nke a na-achọpụta site na kọmputa kọmputa (CT) ka bụ ihe ịma aka na omume ụlọ ọgwụ.N'ebe a, anyị na-akọwapụta metabolome zuru ụwa ọnụ nke ihe nlele serum 480, gụnyere njikwa ahụike, nodules ngụgụ na-adịghị mma, na adenocarcinoma nke akpa ume I.Adenocarcinomas na-egosipụta profaịlụ metabolomic pụrụ iche, ebe nodules na-adịghị mma na ndị nwere ahụike nwere nnukwu myirịta na profaịlụ metabolomic.N'ime otu nchoputa (n = 306), achọpụtara usoro metabolites 27 iji mata ọdịiche dị n'etiti nodules na-adịghị mma na nke na-adịghị mma.AUC nke ụdị ịkpa ókè na nkwado dị n'ime (n = 104) na nkwado mpụga (n = 111) otu bụ 0.915 na 0.945 n'otu n'otu.Nyocha ụzọ gosipụtara mmụba glycolytic metabolites jikọtara ya na mbelata tryptophan na ngụgụ adenocarcinoma serum ma e jiri ya tụnyere nodules na-adịghị mma na njikwa ahụike, ma tụọ aro na iji tryptophan na-akwalite glycolysis na mkpụrụ ndụ kansa akpa ume.Ọmụmụ ihe anyị na-egosipụta uru nke serum metabolite biomarkers na-enyocha ihe ize ndụ nke nodules pulmonary nke CT chọpụtara.
Nchọpụta mbụ dị oke mkpa iji kwalite ọnụego nlanarị maka ndị ọrịa kansa.Nsonaazụ sitere na US National Lung Cancer Screening Trial (NLST) na European NELSON Study egosila na nyocha na obere dose computed tomography (LDCT) nwere ike belata nke ukwuu ọnwụ ọrịa cancer akpa ume na otu dị oke egwu1,2,3.Ebe ọ bụ na eji LDCT eme ihe n'ọtụtụ ebe maka nyocha ọrịa cancer akpa ume, ihe omume nke nchọpụta redio na mberede nke asymptomatic pulmonary nodules nọgidere na-abawanye 4.A kọwapụtara nodules pulmonary dị ka oghere oghere ruo 3 cm na dayameta 5.Anyị na-eche ihe isi ike ihu n'ịtụle ohere nke malignancy na imeri ọnụ ọgụgụ dị ukwuu nke nodules pulmonary achọpụtara na mberede na LDCT.Mmachi nke CT nwere ike iduga nyocha nyocha ugboro ugboro na nsonaazụ ụgha, na-eduga na ntinye aka na-enweghị isi na ọgwụgwọ6.Ya mere, ọ dị mkpa ịzụlite ndị na-ahụ maka ihe ndị a pụrụ ịdabere na ya na ndị bara uru iji chọpụta ọrịa cancer akpa ume n'ụzọ ziri ezi n'oge mmalite ma chọpụta ọtụtụ nodules na-adịghị mma na nchọpụta mbụ 7.
Nnyocha molekụla zuru oke nke ọbara (serum, plasma, sel mononuclear dị n'akụkụ ọbara), gụnyere genomics, proteomics ma ọ bụ DNA methylation8,9,10, emeela ka mmasị na-abawanye na nchọpụta nke ndị na-achọpụta biomarkers maka ọrịa cancer akpa ume.Ka ọ dị ugbu a, metabolomics na-abịaru nso na-atụ ngwaahịa njedebe cellular nke omume endogenous na exogenous na-emetụta ya mere a na-etinye ya n'ọrụ ịkọ mmalite na nsonaazụ ọrịa.Liquid chromatography-tandem mass spectrometry (LC-MS) bụ usoro a na-ejikarị eme ihe maka ọmụmụ metabolomics n'ihi oke uche ya na oke ike dị elu, nke nwere ike kpuchie metabolites nwere ụdị physicochemical dị iche iche11,12,13.Ọ bụ ezie na a na-eji nyocha metabolomic zuru ụwa ọnụ nke plasma / serum iji chọpụta ihe ndị na-emepụta ihe na-ejikọta ya na ọrịa cancer akpa ume 14,15,16,17 na ọgwụgwọ ọgwụgwọ, 18 serum metabolite classifiers iji mara ọdịiche dị n'etiti benign na malignant nodules na-anọgide na-amụba nke ọma.-oke nnyocha.
Adenocarcinoma na squamous cell carcinoma bụ isi ihe abụọ bụ isi nke ọrịa cancer akpa ume na-abụghị obere cell (NSCLC).Nyocha nyocha CT dị iche iche na-egosi na adenocarcinoma bụ ụdị ọrịa cancer akpa ume na-ahụkarị1,19,20,21.N'ime ọmụmụ ihe a, anyị na-eji ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) iji mee nyocha metabolomics na mkpokọta serum 695, gụnyere njikwa ahụike, nodules pulmonary benign, na CT-achọpụtara ≤3 cm.Nyocha maka adenocarcinoma akpa ume Stage I.Anyị chọpụtara otu panel nke metabolites serum nke na-ekewa adenocarcinoma ngụgụ site na nodules na-adịghị mma na njikwa ahụike.Ntụle ịba ụba nke ụzọ ụzọ kpughere na tryptophan na-adịghị mma na metabolism glucose bụ mgbanwe a na-ahụkarị na adenocarcinoma nku ume ma e jiri ya tụnyere nodules na-adịghị mma na njikwa ahụike.N'ikpeazụ, anyị guzobe ma kwadoo serum metabolic classifier nke nwere nkọwa dị elu na nghọta iji mata ọdịiche dị n'etiti ajọ ọrịa nodules na-adịghị mma na nke na-adịghị mma nke LDCT chọtara, nke nwere ike inye aka na nchọpụta mbụ dị iche iche na nyocha ihe ize ndụ.
N'ime ọmụmụ ihe ugbu a, a na-anakọta ihe nlele serum nke mmekọahụ na afọ dakọtara site na njikwa ahụike 174, ndị ọrịa 292 nwere nodules pulmonary na-adịghị mma, yana ndị ọrịa 229 nwere adenocarcinoma nke iku ume I.E gosipụtara njirimara igwe mmadụ nke isiokwu 695 na tebụl mgbakwunye 1.
Dị ka e gosiri na Figure 1a, ngụkọta nke 480 serum samples, gụnyere 174 ahụike njikwa (HC), 170 benign nodules (BN), na 136 stage I lung adenocarcinoma (LA) samples, ka anakọtara na Sun Yat-sen University Cancer Center.Otu nchọta maka profaịlụ metabolomic na-ezubere iche site na iji chromatography mmiri na-arụ ọrụ nke ọma-elu-mkpebi mass spectrometry (UPLC-HRMS).Dị ka egosiri na foto mgbakwunye 1, metabolites dị iche iche dị n'etiti LA na HC, LA na BN ka achọpụtara iji guzobe usoro nhazi ọkwa na n'ihu nyochaa nyocha ụzọ dị iche iche.Ihe nlele 104 nke Sun Yat-sen University Cancer Center chịkọtara na nlele 111 nke ụlọ ọgwụ abụọ ndị ọzọ chịkọtara na nkwado nke ime na mpụga, n'otu n'otu.
Onu ogugu ihe omumu na otu nchoputa nke mere nyocha nke metabolomics zuru ụwa ọnụ site na iji ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).Ntụle ịkpa oke n'akụkụ akụkụ pere mpe (PLS-DA) nke mkpokọta metabolome nke 480 serum sample sitere na otu ọmụmụ, gụnyere njikwa ahụike (HC, n = 174), nodules benign (BN, n = 170), na ogbo I nku ume adenocarcinoma (Los Angeles, n = 136).+ ESI, ọnọdụ ionization electrospray ziri ezi, -ESI, ọnọdụ ionization electrospray adịghị mma.c–e Metabolites nwere oke dị iche iche dị iche iche na otu abụọ enyere aka (nleba anya Wilcoxon nwere ogologo abụọ, ọnụego nchoputa ụgha gbanwere p uru, FDR <0.05) na-egosi na ọbara ọbara (mgbanwe mpịachi> 1.2) na acha anụnụ anụnụ (mgbanwe mkpụkọ <0.83) .) egosiri na eserese mgbawa ugwu.f Maapụ ikpokọta okpomọkụ nke usoro na-egosi ọdịiche dị ukwuu na ọnụọgụ metabolites akọwapụtara n'etiti LA na BN.A na-enye data isi mmalite n'ụdị faịlụ data isi mmalite.
Mkpokọta metabolome serum nke 174 HC, 170 BN na 136 LA n'ime otu nchọpụta ka ejiri nyocha UPLC-HRMS nyochaa.Anyị na-ebu ụzọ gosi na nlebanya njikwa mma (QC) na-agbakọta nke ọma n'etiti usoro nyocha akụrụngwa nke anaghị elekọta (PCA), na-akwado nkwụsi ike nke arụmọrụ ọmụmụ ugbu a (Ntinye 2).
Dị ka e gosiri na ele mmadụ anya n'ihu na-akpachapụ anya analysis (PLS-DA) na Figure 1 b, anyị chọpụtara na e nwere doro anya ọdịiche dị n'etiti LA na BN, LA na HC na nti (+ ESI) na ọjọọ (-ESI) electrospray ionization ụdịdị. .dịpụrụ adịpụ.Agbanyeghị, enweghị nnukwu ọdịiche dị n'etiti BN na HC na ọnọdụ +ESI na -ESI.
Anyị chọtara 382 dị iche iche dị iche iche n'etiti LA na HC, 231 dị iche iche dị n'etiti LA na BN, na 95 dị iche iche dị iche iche n'etiti BN na HC (Wilcoxon bịanyere aka n'akwụkwọ ule, FDR <0.05 na otutu mgbanwe> 1.2 ma ọ bụ <0.83) (Figure .1c-e). )..A kọwakwuru ọnụ ọgụgụ kachasị elu (Nkọwa Data 3) megide nchekwa data (mzCloud/HMDB/Chemspider Library) site na uru m/z, oge njide na nchọta ihe nrịba ama nkewa (nkọwa akọwapụtara na ngalaba Ụzọ) 22 .N'ikpeazụ, 33 na 38 nkọwa metabolites nwere nnukwu ọdịiche dị n'ụba ka achọpụtara maka LA vesos BN (Figure 1f and Supplementary Table 2) na LA vesos HC (Mgbakwunye 3 na Tebụl Mgbakwunye 2), n'otu n'otu.N'ụzọ dị iche, ọ bụ naanị metabolites 3 nwere nnukwu ọdịiche dị n'ụba ka achọpụtara na BN na HC (Table 2 na mgbakwunye), kwekọrọ na ndakọrịta n'etiti BN na HC na PLS-DA.Metabolites ndị a dị iche na-ekpuchi ọtụtụ ụdị biochemicals (Nkọwapụta foto 4).N'ịchịkọta ọnụ, nsonaazụ ndị a na-egosipụta mgbanwe dị ịrịba ama na serum metabolome nke na-egosipụta mgbanwe ọjọọ nke ọrịa cancer akpa ume n'oge mbụ ma e jiri ya tụnyere nodules na-adịghị mma ma ọ bụ ihe ndị nwere ahụike.Ka ọ dị ugbu a, myirịta nke metabolome serum nke BN na HC na-egosi na nodules na-adịghị mma nwere ike ịkọrọ ndị nwere ahụike ọtụtụ njirimara ndu.Nyere na mmụgharị mkpụrụ ndụ ihe na-eto eto epidermal (EGFR) na-adịkarị na ngụgụ adenocarcinoma subtype 23, anyị chọrọ ịchọpụta mmetụta mmụgharị ndị ọkwọ ụgbọ ala na serum metabolome.Anyị wee nyochaa mkpokọta metabolomic profaịlụ nke ikpe 72 nwere ọnọdụ EGFR na otu adenocarcinoma ngụgụ.N'ụzọ na-akpali mmasị, anyị chọtara profaịlụ atụnyere n'etiti ndị ọrịa EGFR mutant (n = 41) na ndị ọrịa anụ ọhịa EGFR (n = 31) na nyocha PCA (Nkọwa Mgbakwunye 5a).Otú ọ dị, anyị chọpụtara metabolites 7 nke ụbara ya gbanwere nke ukwuu na ndị ọrịa nwere mgbanwe EGFR ma e jiri ya tụnyere ndị ọrịa nwere ụdị EGFR anụ ọhịa (t ule, p <0.05 na mpịachi mgbanwe> 1.2 ma ọ bụ <0.83) (Ntụpọ 5b).Ọtụtụ n'ime metabolites ndị a (5 n'ime 7) bụ acylcarnitines, nke na-arụ ọrụ dị mkpa na ụzọ oxidation fatty acid.
Dị ka e gosipụtara na usoro ọrụ egosiri na Figure 2 a, enwetara biomarkers maka nhazi ọkwa nodule site na iji ndị na-arụ ọrụ nkwụsịtụ zuru oke na nhọrọ dabere na metabolites 33 dị iche iche akọwapụtara na LA (n = 136) na BN (n = 170).Ngwakọta kacha mma nke mgbanwe (LASSO) - ọnụọgụ abụọ logistic ụdị regression.A na-eji nkwenye cross-ugboro iri iji nwalee ntụkwasị obi nke ihe nlereanya ahụ.A na-edozi nhọrọ dịgasị iche iche na nhazi nke oke site na oke ntaramahụhụ nwere ike iji oke λ24.A na-eme nyocha nke metabolomics zuru ụwa ọnụ n'adabereghị na nkwenye dị n'ime (n = 104) na nkwado mpụga (n = 111) otu iji nwalee arụmọrụ nhazi nke ụdị ịkpa ókè.N'ihi ya, 27 metabolites na nchọpụta nchọpụta achọpụtara dị ka ụdị ịkpa ókè kachasị mma nke nwere uru AUC kachasị ukwuu (Fig 2b), n'ime nke 9 nwere ụbara ọrụ na 18 na-ebelata ọrụ na LA ma e jiri ya tụnyere BN (Fig 2c).
Nrụ ọrụ maka iwulite ọkwa ọkwa nodule pulmonary, gụnyere ịhọrọ panel kacha mma nke serum metabolites na nchọta nchoputa site na iji usoro nkwụghachi azụ nke ọnụọgụ abụọ site na nkwenye okpukpu iri na ịtụle arụmọrụ amụma na ntinye nkwado nke ime na mpụga.b Ọnụọgụ nkwado nke LASSO regression model maka nhọpụta biomarker metabolic.Ọnụọgụ ndị enyere n'elu na-anọchi anya nkezi ọnụọgụ biomarkers ahọpụtara na λ enyere.Ahịrị nwere ntụpọ uhie na-anọchite anya nkezi uru AUC na lambda kwekọrọ.Ogwe njehie isi awọ na-anọchi anya ụkpụrụ AUC kacha nta na kacha.Ahịrị nwere ntụpọ na-egosi ihe nlereanya kachasị mma yana ndị 27 ahọpụtara biomarkers.AUC, mpaghara n'okpuru njirimara na-arụ ọrụ nnata (ROC).c Gbanwee mgbanwe nke metabolites 27 ahọpụtara n'ime otu LA ma e jiri ya tụnyere ndị otu BN na otu nchọpụta.Ogidi uhie - ịgbalite.Kọlụm na-acha anụnụ anụnụ bụ ọdịda.d–f akụkụ nke ndị nnata na-arụ ọrụ (ROC) na-egosi ike nke ụdị ịkpa oke dabere na nchikota metabolite 27 na nchọta, n'ime, na mpụta nkwado.A na-enye data isi mmalite n'ụdị faịlụ data isi mmalite.
Emepụtara ihe atụ amụma dabere na ọnụọgụ nkwụghachi azụ dị arọ nke metabolites 27 ndị a (Table 3 mgbakwunye).Nyocha ROC dabere na metabolites 27 ndị a na-ewepụta mpaghara n'okpuru ọnụọgụ (AUC) uru nke 0.933, mmetụta nke otu nchọpụta bụ 0.868, na nkọwapụta bụ 0.859 (Fig 2d).Ka ọ dị ugbu a, n'ime metabolites dị iche iche nke 38 akọwapụtara n'etiti LA na HC, setịpụ metabolites 16 nwetara AUC nke 0.902 nwere uche nke 0.801 na nkọwapụta nke 0.856 na ịkpa ókè LA sitere na HC (Ngbakwunye 6a-c).A na-atụnyere ụkpụrụ AUC dabere na mpịachi dị iche iche na-agbanwe ụzọ maka metabolites dị iche iche.Anyị chọpụtara na ụdị nhazi ọkwa ahụ rụrụ nke ọma n'ịkpa oke n'etiti LA na BN (HC) mgbe ọkwa mgbanwe mpịaji ka atọrọ ka ọ bụrụ 1.2 na 1.5 ma ọ bụ 2.0 (Ọnọdụ mgbakwunye 7a,b).Ụdị nhazi ọkwa ahụ, dabere na otu metabolite 27, ka akwadoro n'ime otu n'ime na mpụga.AUC bụ 0.915 (ntụgharị uche 0.867, nkọwa 0.811) maka nkwenye n'ime na 0.945 (mmetụta 0.810, kpọmkwem 0.979) maka nkwenye mpụga (Fig. 2e, f).Iji nyochaa arụmọrụ interlaboratory, a na-enyocha ihe nlele 40 sitere na ndị otu mpụga na ụlọ nyocha dị n'èzí dịka akọwara na ngalaba Ụzọ.Izi ezi nhazi ọkwa nwetara AUC nke 0.925 (Ntụpọ mgbakwunye 8).N'ihi na carcinoma akpa ume squamous cell (LUSC) bụ ụdị nke abụọ a na-ahụkarị nke ọrịa cancer akpa ume na-abụghị obere cell (NSCLC) na-esote adenocarcinoma ume ume (LUAD), anyị nwalekwara ike ikike nke profaịlụ metabolic.BN na ikpe 16 nke LUSC.AUC nke ịkpa oke n'etiti LUSC na BN bụ 0.776 (Nkọwa mgbakwunye 9), na-egosi ike adịghị mma ma e jiri ya tụnyere ịkpa ókè n'etiti LUAD na BN.
Nnyocha e mere egosiwo na nha nodules na ihe oyiyi CT na-ejikọta ya na ohere nke malignancy ma nọgide na-abụ isi ihe na-achọpụta ọgwụgwọ nodule25,26,27.Nyocha nke data sitere na nnukwu ìgwè nke nyocha nyocha NELSON gosiri na ihe ize ndụ nke malignancy na isiokwu ndị nwere ọnụ <5 mm ọbụna yiri nke ahụ na isiokwu ndị na-enweghị ọnụ 28.Ya mere, nke kacha nta nke chọrọ nlekota CT mgbe niile bụ 5 mm, dị ka British Thoracic Society (BTS) kwadoro, na 6 mm, dị ka Fleischner Society 29 kwadoro.Agbanyeghị, nodules buru ibu karịa 6 mm na enweghị njiri mara mma doro anya, nke a na-akpọ indeterminate pulmonary nodules (IPN), ka bụ nnukwu ihe ịma aka na nyocha na njikwa na omume ụlọ ọgwụ30,31.Anyị nyochara ma nha nodule ọ na-emetụta mbinye aka metabolomic site na iji ihe atụ agbakọtara site na nchọta na nkwado n'ime.Na-elekwasị anya na biomarkers 27 kwadoro, anyị buru ụzọ tulee profaịlụ PCA nke HC na BN sub-6 mm metabolomes.Anyị chọpụtara na ọtụtụ n'ime data data maka HC na BN overlapped, na-egosi na serum metabolite ọkwa dị na abụọ otu (Fig. 3a).Maapụ atụmatụ n'ofe oke nha dị iche iche ka echekwara na BN na LA (Fig 3b, c), ebe a na-ahụ nkewa n'etiti nodules na-adịghị mma na nke na-adịghị mma na 6-20 mm (Fig 3d).Ndị otu a nwere AUC nke 0.927, nkọwa nke 0.868, na mmetụta nke 0.820 maka ịkọ nkọ nke nodules na-atụle 6 ruo 20 mm (Fig 3e, f).Nsonaazụ anyị na-egosi na classifier nwere ike weghara mgbanwe metabolic kpatara ngbanwe dị njọ n'oge, n'agbanyeghị nha nodule.
Ntụle profaịlụ PCA n'etiti otu ndị akọwapụtara dabere na nkewa metabolic nke metabolites 27.CC na BN <6 mm.b BN <6 mm vs BN 6–20 mm.na LA 6–20 mm megide LA 20–30 mm.g BN 6–20 mm na 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 Ejiji njirimara na-arụ ọrụ nke nnata (ROC) na-egosi arụmọrụ ụdị ịkpa ókè maka nodules 6-20 mm.f A na-agbakọ ụkpụrụ nke puru omume dabere na ihe ngbanwe nke mgbagha maka nodules na-atụ 6-20 mm.Ahịrị ntụpọ isi awọ na-anọchi anya uru mbelata kacha mma (0.455).Ọnụọgụ ndị dị n'elu na-anọchi anya pasenti ikpe ndị a tụrụ anya maka Los Angeles.Jiri ọdụ abụọ nke Student t ule.PCA, isi akụrụngwa nyocha.Mpaghara AUC n'okpuru usoro.A na-enye data isi mmalite n'ụdị faịlụ data isi mmalite.
A na-ahọrọ ihe atụ anọ (afọ 44-61) nwere ụdị nodule pulmonary (7-9 mm) iji gosipụta arụmọrụ nke ụdị amụma amụma ọjọọ (Fig. 4a, b).Na nyocha mbụ, Case 1 gosipụtara dị ka nodule siri ike nwere calcification, njirimara jikọtara ya na ịdị mma, ebe ikpe 2 gosipụtara dị ka nodule na-enweghị njedebe siri ike na-enweghị njiri mara mma doro anya.Usoro atọ nke nyocha CT na-esochi gosipụtara na ikpe ndị a nọgidere na-adịgide adịgide n'ime afọ 4 ma ya mere a na-ewere ya dị ka nodules na-adịghị mma (Fig 4a).E jiri ya tụnyere nyocha ụlọ ọgwụ nke nyocha CT, otu-shot serum metabolite analysis na ụdị nkewa nke ugbu a ngwa ngwa ma chọpụta nke ọma nodules ndị a na-adịghị mma dabere na mgbochi puru omume (Table 1).Ọgụgụ 4b ma ọ bụrụ na 3 na-egosi nodule nwere akara nke mgbagha azụ, nke a na-ejikọkarị ya na malignancy32.Ikpe 4 gosipụtara dị ka nodule na-enweghị njedebe na-enweghị ihe akaebe na-egosi ihe kpatara adịghị mma.Aburu amụma ikpe ndị a niile dị ka ndị na-adịghị mma dịka ụdị nhazi ọkwa (Table 1).E gosipụtara ntule nke adenocarcinoma nku ume site na nyocha histopathological mgbe ịwa ahụ nku ume (Fig 4b).Maka ntọala nkwado nke mpụga, onye na-ahụ maka metabolic buru amụma n'ụzọ ziri ezi okwu abụọ nke nodules ngụgụ na-anaghị apụta nke kariri 6 mm (Ngbakwunye 10).
Ihe onyonyo CT nke windo axial nke ngụgụ nke ikpe abụọ nke nodules benign.N'ihe banyere 1, CT scan mgbe afọ 4 gachara gosipụtara nodule siri ike siri ike na-atụ 7 mm na calcification na lobe ala aka nri.N'ihe banyere 2, CT scan mgbe afọ 5 gachara kpughere nodule kwụsiri ike, nke nwere akụkụ siri ike nke nwere dayameta nke 7 mm na lobe nke aka nri.b Axial window CT onyogho nke ngụgụ na kwekọrọ pathological ọmụmụ nke abụọ nke ogbo I adenocarcinoma tupu nku ume resection.Ikpe 3 kpughere nodule nke nwere dayameta nke 8 mm n'akụkụ aka nri nke elu nke nwere mgbagha pleural.Ikpe 4 kpughere nodule glass ala siri ike na-atụ 9 mm na lobe nke elu aka ekpe.Hematoxylin na eosin (H&E) ntanye nke anụ ahụ resected resected (akara mmanya = 50 μm) na-egosi usoro uto acinar nke nku ume adenocarcinoma.Àkú na-egosi nodules achọpụtara na foto CT.Onyonyo H&E bụ onyonyo nnochite anya nke otutu (> 3) obere oghere nke onye na-ahụ maka ọrịa nwalere.
N'ịchịkọta ọnụ, nsonaazụ anyị na-egosi uru nke serum metabolite biomarkers na nchọpụta dị iche iche nke nodules pulmonary, nke nwere ike ịkpata ihe ịma aka mgbe ị na-enyocha nyocha CT.
Dabere na panel metabolite dị iche, anyị chọrọ ịchọpụta ihe jikọrọ ndụ nke nnukwu mgbanwe metabolic.Ntụle mmụba nke ụzọ KEGG site na MetaboAnalyst chọpụtara ụzọ 6 na-agbanwe agbanwe nke ukwuu n'etiti otu abụọ enyere (LA vs. HC na LA vs. BN, gbanwee p ≤ 0.001, mmetụta> 0.01).Mgbanwe ndị a bụ ihe mgbagwoju anya na pyruvate metabolism, tryptophan metabolism, niacin na nicotinamide metabolism, glycolysis, TCA okirikiri, na purine metabolism (Fig. 5a).Anyị gakwara n'ihu rụpụta metabolomics ezubere iche iji nyochaa nnukwu mgbanwe site na iji ọnụọgụ zuru oke.Mkpebi nke metabolites nkịtị n'ụzọ ndị a na-agbanwekarị site na spectrometry quadrupole mass spectrometry atọ (QQQ) na-eji ezigbo metabolite.A na-agụnye njirimara ọnụọgụgụ nke metabolomics ihe ọmụmụ ihe atụ na tebụl mgbakwunye 4. N'ikwekọ na nsonaazụ metabolomics zuru ụwa ọnụ anyị, nyocha nke quantitative kwadoro na hypoxanthine na xanthine, pyruvate, na lactate mụbara na LA ma e jiri ya tụnyere BN na HC (Fig 5b, c. p <0.05).Agbanyeghị, ọ nweghị nnukwu ọdịiche dị na metabolites ndị a ka ahụrụ n'etiti BN na HC.
Nyocha nkwalite ụzọ KEGG nke metabolites dị iche iche dị na LA otu ma e jiri ya tụnyere ndị otu BN na HC.A na-eji Globaltest ọdụdụ abụọ mee ihe, a na-edozi ụkpụrụ p site na iji usoro Holm-Bonferroni (gbanwee p ≤ 0.001 na nha mmetụta> 0.01).b-d violin na-egosi hypoxanthine, xanthine, lactate, pyruvate, na ọkwa tryptophan na serum HC, BN, na LA nke LC-MS / MS (n = 70 kwa otu).Ahịrị nwere ntụpọ ọcha na ojii na-egosi etiti na nke anọ, n'otu n'otu.e violin ibé na-egosi normalized Log2TPM (nsụgharị kwa nde) mRNA okwu nke SLC7A5 na QPRT na nku ume adenocarcinoma (n = 513) tụnyere nkịtị ngụgụ anụ ahụ (n = 59) na LUAD-TCGA dataset.Igbe na-acha ọcha na-anọchi anya etiti etiti, ahịrị ojii kwụ n'ahịrị na etiti na-anọchi anya etiti, na ahịrị ojii kwụ ọtọ na-esi na igbe ahụ na-anọchi anya oge ntụkwasị obi 95% (CI).f Pearson mmekorita atụmatụ nke SLC7A5 na GAPDH okwu na nku ume adenocarcinoma (n = 513) na nkịtị akpa ume anụ ahụ (n = 59) na TCGA dataset.Mpaghara isi awọ na-anọchite anya 95% CI.r, Pearson njikọ ọnụ ọgụgụ.g Ọkwa tryptophan cell ahazigharịrị n'ime sel A549 ejiri njikwa shRNA na-enweghị isi (NC) na shSLC7A5 (Sh1, Sh2) kpebiri site na LC-MS/MS.A na-ewepụta nyocha ọnụ ọgụgụ nke ihe atụ ise nwere onwe ha n'otu ọ bụla.h Ọkwa sel nke NADt (ngụkọta NAD, gụnyere NAD + na NADH) na sel A549 (NC) na SLC7A5 knockdown A549 sel (Sh1, Sh2).A na-ewepụta nyocha ọnụ ọgụgụ nke ihe atụ atọ nwere onwe ha n'ime otu ọ bụla.A na-atụ ọrụ Glycolytic nke mkpụrụ ndụ A549 tupu na mgbe SLC7A5 knockdown tụrụ site na extracellular acidification rate (ECAR) (n = 4 ụdị nnwere onwe nke ndu kwa otu).2-DG,2-deoxy-D-glucose.Ejiri ule nwa akwụkwọ nwere ọdụ abụọ na (b–h).Na (g-i), mpe mpe mpe mpe akwa na-anọchi anya ± SD, nnwale ọ bụla emere ugboro atọ n'adabereghị na nsonaazụ ya yiri ya.A na-enye data isi mmalite n'ụdị faịlụ data isi mmalite.
N'ịtụle mmetụta dị ịrịba ama nke mgbanwe tryptophan metabolism na otu LA, anyị tụlekwara ọkwa serum tryptophan na HC, BN, na LA otu site na iji QQQ.Anyị chọpụtara na serum tryptophan belatara na LA ma e jiri ya tụnyere HC ma ọ bụ BN (p <0.001, Figure 5d), nke kwekọrọ na nchoputa gara aga na-ekesa ọkwa tryptophan dị ala na ndị ọrịa nwere ọrịa cancer akpa ume karịa na njikwa ahụike sitere na njikwa njikwa33,34. ,35.Nnyocha ọzọ na-eji PET/CT tracer 11C-methyl-L-tryptophan chọpụtara na oge njigide mgbaàmà tryptophan na anụ ahụ cancer akpa ume mụbara nke ukwuu ma e jiri ya tụnyere ọnya na-adịghị mma ma ọ bụ anụ ahụ nkịtị36.Anyị na-eche na mbelata nke tryptophan na serum LA nwere ike igosipụta nnabata tryptophan na-arụ ọrụ site na mkpụrụ ndụ kansa akpa ume.
A makwaara na ngwaahịa njedebe nke ụzọ kynurenine nke tryptophan catabolism bụ NAD + 37,38, nke bụ ihe dị mkpa maka mmeghachi omume nke glyceraldehyde-3-phosphate na 1,3-bisphosphoglycerate na glycolysis39.Ọ bụ ezie na ọmụmụ ihe gara aga lekwasịrị anya na ọrụ nke tryptophan catabolism na nhazi usoro mgbochi, anyị chọrọ ịkọwapụta mmekọrịta dị n'etiti dysregulation tryptophan na ụzọ glycolytic a hụrụ na ọmụmụ ihe ugbu a.Solute transporter ezinụlọ 7 onye 5 (SLC7A5) ka amara na ọ bụ onye na-ebugharị tryptophan43,44,45.Quinolinic acid phosphoribosyltransferase (QPRT) bụ enzyme nke dị na ala nke ụzọ kynurenine nke na-atụgharị quinolinic acid na NAMN46.Nyocha nke dataset LUAD TCGA gosiri na ma SLC7A5 na QPRT na-emeziwanye nke ọma na anụ ahụ tumor ma e jiri ya tụnyere anụ ahụ nkịtị (Fig 5e).A hụrụ mmụba a n'ọkwa nke mbụ na nke abụọ yana ọkwa III na IV nke adenocarcinoma nku ume (Ntinye mgbakwunye 11), na-egosi ọgba aghara n'oge na metabolism tryptophan jikọtara ya na tumorigenesis.
Na mgbakwunye, ihe ndekọ data LUAD-TCGA gosipụtara mmekọrịta dị mma n'etiti SLC7A5 na GAPDH mRNA okwu na nlele ndị ọrịa kansa (r = 0.45, p = 1.55E-26, Figure 5f).N'ụzọ dị iche, ọnweghị njikọ dị ịrịba ama n'etiti mbinye aka mkpụrụ ndụ ihe nketa dị na anụ ahụ nkịtị (r = 0.25, p = 0.06, Figure 5f).Knockdown nke SLC7A5 (Ngbakwunye 12) na sel A549 belatara nke ukwuu tryptophan cellular na NAD (H) ọkwa (Njirimara 5g,h), na-ebute ọrụ glycolytic attenuated dị ka atụpụtara site na extracellular acidification ọnụego (ECAR) (Nyocha 1).5i).Ya mere, dabere na mgbanwe metabolic na serum na nchọpụta in vitro, anyị na-eche na metabolism tryptophan nwere ike ịmepụta NAD + site na ụzọ kynurenine ma rụọ ọrụ dị mkpa n'ịkwalite glycolysis na ọrịa cancer akpa ume.
Nnyocha e mere egosiwo na ọnụ ọgụgụ dị ukwuu nke nodules na-adịghị ahụ anya nke LDCT chọpụtara nwere ike ime ka ọ dị mkpa maka nyocha ọzọ dị ka PET-CT, lung biopsy, na overtreatment n'ihi nchọpụta ụgha na-adịghị mma nke malignancy.31 Dị ka e gosiri na Figure 6. Ọmụmụ ihe anyị chọpụtara otu panel nke metabolites serum nwere uru nchoputa nke nwere ike imeziwanye ihe ize ndụ stratification na nlekọta na-esote nke nodules pulmonary nke CT chọpụtara.
A na-enyocha nodules pulmonary site na iji tomography gbakọọ dị ala (LDCT) nwere njirimara onyonyo nke na-egosi ihe ndị na-adịghị mma ma ọ bụ njọ.Nsonaazụ ejighị n'aka nke nodules nwere ike iduga na nleta na-esochi ya ugboro ugboro, ntinye aka na-adịghị mkpa, na ọgwụgwọ.Ntinye nke serum metabolic classifiers nwere uru diagnostic nwere ike melite ntule ihe ize ndụ na nlekọta na-esote nke nodules pulmonary.PET positron emission tomography.
Data sitere na US NLST ọmụmụ na European NELSON ọmụmụ na-atụ aro na nyocha otu ndị nwere nnukwu ihe ize ndụ nwere obere ihe mgbagwoju anya (LDCT) nwere ike ibelata ọrịa cancer akpa ume1,3.Agbanyeghị, ntule ihe egwu na nlekọta ụlọ ọgwụ na-esote nke ọnụ ọgụgụ buru ibu nke nodules akpa ume mberede nke LDCT chọpụtara ka bụ ihe siri ike.Ebumnuche bụ isi bụ ịkwalite nhazi ọkwa ziri ezi nke ụkpụrụ LDCT dị adị site na itinye ndị na-ahụ maka biomarkers a pụrụ ịdabere na ya.
Achọpụtara ụfọdụ ihe ndị na-ahụ maka ihe ndị dị ndụ, dị ka metabolites ọbara, site n'iji ọrịa cancer akpa ume tụnyere njikwa ahụike15,17.N'ime ọmụmụ ihe ugbu a, anyị lekwasịrị anya na ntinye nyocha nke metabolomics serum iji mata ọdịiche dị n'etiti nodules pulmonary na-adịghị mma na nke na-adịghị mma nke LDCT chọpụtara na mberede.Anyị tụlere metabolome zuru ụwa ọnụ nke njikwa ahụike (HC), nodules nku ume na-adịghị mma (BN), na ọkwa I nku ume adenocarcinoma (LA) site na iji nyocha UPLC-HRMS.Anyị chọpụtara na HC na BN nwere profaịlụ metabolic yiri nke ahụ, ebe LA gosipụtara mgbanwe dị ịrịba ama ma e jiri ya tụnyere HC na BN.Anyị chọpụtara usoro abụọ nke serum metabolites nke dị iche LA na HC na BN.
Atụmatụ njirimara dabere na LDCT dị ugbu a maka nodules na-adịghị mma na nke na-adịghị mma na-adabere na nha, njupụta, morphology na ọnụego uto nke nodules karịa oge 30.Nnyocha ndị gara aga egosila na oke nodules nwere njikọ chiri anya na ohere nke ọrịa cancer akpa ume.Ọbụna na ndị ọrịa nwere nnukwu ihe ize ndụ, ihe ize ndụ nke malignancy na ọnụ <6 mm bụ <1%.Ihe ize ndụ nke malignancy maka nodules na-atụ 6 ruo 20 mm sitere na 8% ruo 64% 30.Ya mere, Fleischner Society na-atụ aro ka a na-ebipụ dayameta nke 6 mm maka nlekota CT na-aga n'ihu.29 Otú ọ dị, nleba anya na njikwa ihe ize ndụ nke nodules pulmonary pulmonary (IPN) nke karịrị 6 mm emebeghị nke ọma 31.Nlekọta ọrịa obi nke afọ ime ugbu a na-adaberekarị na nchere nche yana nlekota CT ugboro ugboro.
Dabere na metabolome emelitere, anyị gosipụtara na nke mbụ nchikota nke mbinye aka metabolomic n'etiti ndị ahụ siri ike na nodules na-adịghị mma <6 mm.Myirịta dị ndụ kwekọrọ na nchọpụta CT gara aga na ihe ize ndụ nke malignancy maka nodules <6 mm dị ala dị ka isiokwu na-enweghị ọnụ.30 Ekwesịrị iburu n'uche na nsonaazụ anyị na-egosikwa na nodules na-adịghị mma <6 mm na ≥6 mm nwere elu. myirịta na profaịlụ metabolomic, na-atụ aro na nkọwa ọrụ nke etiology benign na-agbanwe agbanwe n'agbanyeghị nha nodule.N'ihi ya, nke oge a diagnostic serum metabolite panels nwere ike inye otu nyocha dị ka a na-achị achị mgbe achọpụtara nodules na mbụ na CT ma nwee ike ibelata nlekota nlekota.N'otu oge ahụ, otu panel nke metabolic biomarkers na-amata ọdịiche dị njọ nke nodules ≥6 mm n'ogo site na nodules benign ma nye amụma ziri ezi maka IPN nke nha nha na njirimara morphological na-enweghị atụ na ihe oyiyi CT.Nke a serum metabolism classifier rụrụ nke ọma n'ịkọ amụma ọjọọ nke nodules ≥6 mm na AUC nke 0.927.N'ịchịkọta ọnụ, nsonaazụ anyị na-egosi na mbinye aka metabolomic serum pụrụ iche nwere ike igosipụta mgbanwe metabolic mbụ nke etuto na-ebute ma nwee uru dị ka ndị na-ebu amụma ihe egwu, na-adabereghị na nha nodule.
N'ụzọ doro anya, adenocarcinoma ngụgụ (LUAD) na squamous cell carcinoma (LUSC) bụ isi ụdị ọrịa cancer akpa ume na-abụghị obere cell (NSCLC).Nyere na LUSC nwere njikọ siri ike na ụtaba47 na LUAD bụ akụkọ ihe mere eme nke nodules ngụgụ na mberede achọpụtara na CT screening48, e wuru ụdị klas anyị nke ọma maka nlele adenocarcinoma I.Wang na ndị ọrụ ibe lekwasịrị anya na LUAD wee chọpụta mbinye aka lipid itoolu site na iji lipidomics ịmata ọdịiche dị n'ọrịa kansa akpa ume n'oge na ndị nwere ahụike17.Anyị nwalere ụdị nkewa ugbu a na ikpe 16 nke ogbo I LUSC na 74 nodules benign ma hụ izi ezi amụma LUSC dị ala (AUC 0.776), na-atụ aro na LUAD na LUSC nwere ike ịnwe mbinye aka metabolomic nke ha.N'ezie, egosiri LUAD na LUSC dị iche na etiology, mmalite ndu na mkpụrụ ndụ ihe nketa49.Ya mere, ụdị histology ndị ọzọ kwesịrị ịgụnye n'ụdị ọzụzụ maka nchọpụta ndị mmadụ na-achọpụta ọrịa cancer akpa ume na mmemme nyocha.
N'ebe a, anyị chọpụtara ụzọ isii a na-agbanwekarị na adenocarcinoma ngụgụ ma e jiri ya tụnyere njikwa ahụike na nodules na-adịghị mma.Xanthine na hypoxanthine bụ metabolites nkịtị nke ụzọ metabolic purine.N'ikwekọ na nsonaazụ anyị, ndị etiti jikọtara ya na metabolism purine na-abawanye nke ukwuu na serum ma ọ bụ anụ ahụ nke ndị ọrịa nwere adenocarcinoma nku ume ma e jiri ya tụnyere njikwa ahụike ma ọ bụ ndị ọrịa na preinvasive stage15,50.Serum xanthine dị elu na ọkwa hypoxanthine nwere ike igosipụta anabolism nke mkpụrụ ndụ kansa na-ebuwanye ngwa ngwa chọrọ.Dysregulation nke glucose metabolism bụ ihe ama ama nke ọrịa ọrịa kansa 51.N'ebe a, anyị hụrụ mmụba dị ukwuu na pyruvate na lactate na LA otu ma e jiri ya tụnyere HC na BN otu, nke kwekọrọ na akụkọ ndị gara aga nke glycolytic ụzọ abnormalities na serum metabolome profaịlụ nke ndị ọrịa na-abụghị obere cell lung cancer (NSCLC) njikwa ahụike.nsonaazụ ya na-agbanwe agbanwe52,53.
N'ụzọ dị mkpa, anyị hụrụ njikọ dị n'etiti pyruvate na tryptophan metabolism na serum adenocarcinomas ngụgụ.A na-ebelata ọkwa nke tryptophan serum na otu LA ma e jiri ya tụnyere otu HC ma ọ bụ BN.N'ụzọ na-akpali mmasị, ọmụmụ ihe buru ibu gara aga site na iji otu ndị na-atụ anya na-achọpụta na ọnụ ọgụgụ dị ala nke na-ekesa tryptophan na-ejikọta ya na ihe ize ndụ nke ọrịa cancer akpa ume 54.Tryptophan bụ amino acid dị mkpa nke anyị na-enweta kpamkpam site na nri.Anyị na-ekwubi na mbelata serum tryptophan na nku ume adenocarcinoma nwere ike igosi mbelata ngwa ngwa nke metabolite a.A maara nke ọma na njedebe ngwaahịa nke tryptophan catabolism site na ụzọ kynurenine bụ isi iyi nke de novo NAD + njikọ.N'ihi na a na-emepụta NAD + site na ụzọ mgbapụta, mkpa NAD + dị na tryptophan metabolism na ahụike na ọrịa ka ga-ekpebi46.Nyocha anyị nke nchekwa data TCGA gosiri na nkwupụta nke tryptophan transporter solute transporter 7A5 (SLC7A5) abawanyela nke ukwuu na adenocarcinoma nku ume ma e jiri ya tụnyere njikwa nkịtị ma na-ejikọta ya na nkwupụta nke glycolytic enzyme GAPDH.Ọmụmụ ihe ọmụmụ gara aga lekwasịrị anya na ọrụ tryptophan catabolism na-egbochi nzaghachi mgbochi ọrịa antitumor40,41,42.N'ebe a, anyị na-egosi na mgbochi nke tryptophan nnabata site na ịkụda SLC7A5 na mkpụrụ ndụ cancer akpa ume na-ebute mbelata na-esote ọkwa NAD cellular yana mbelata ọrụ glycolytic.Na nchịkọta, ọmụmụ ihe anyị na-enye ihe ndabere ndu maka mgbanwe na serum metabolism jikọtara ya na mgbanwe dị njọ nke adenocarcinoma nku ume.
Mgbanwe EGFR bụ mgbanwe ndị ọkwọ ụgbọ ala na-ahụkarị na ndị ọrịa nwere NSCLC.N'ime ọmụmụ anyị, anyị chọpụtara na ndị ọrịa nwere EGFR mutation (n = 41) nwere profaịlụ metabolomic n'ozuzu dịka ndị ọrịa nwere ụdị anụ ọhịa EGFR (n = 31), n'agbanyeghị na anyị hụrụ mbelata ọbara ọbara nke ụfọdụ ndị ọrịa EGFR mutant na ndị ọrịa acylcarnitine.Ọrụ guzosiri ike nke acylcarnitines bụ ibufe ndị otu acyl site na cytoplasm n'ime matrix mitochondrial, na-eduga na oxidation nke fatty acids iji mepụta ume 55.N'ikwekọ na nchoputa anyị, nnyocha e mere n'oge na-adịbeghị anya chọpụtakwara profaịlụ metabolome dị n'etiti EGFR mutant na EGFR ụdị anụ ahụ site na nyochaa metabolome zuru ụwa ọnụ nke 102 lung adenocarcinoma tissue samples50.N'ụzọ na-akpali mmasị, a hụkwara ọdịnaya acylcarnitine n'ime otu EGFR mutant.Ya mere, ma mgbanwe na ọkwa acylcarnitine na-egosipụta mgbanwe mgbanwe nke EGFR na-akpata na usoro mkpụrụ ndụ ihe nketa nwere ike ịkwado ọmụmụ ihe ọzọ.
N'ikpeazụ, ọmụmụ ihe anyị na-eguzobe serum metabolic classifier maka nchọpụta dị iche iche nke nodules pulmonary ma na-atụ aro usoro ọrụ nke nwere ike ịkwalite nyocha ihe ize ndụ na ịkwado nlekọta ahụike dabere na nyocha CT.
Ndị Kọmitii Ethics nke Sun Yat-sen University Cancer Hospital kwadoro ọmụmụ ihe a, Ụlọ Ọgwụ Njikọ Mbụ nke Mahadum Sun Yat-sen, na Kọmitii Omume nke Ụlọ Ọgwụ Ọrịa Cancer nke Zhengzhou University.Na nchoputa na otu nkwado nke ime, 174 sera sitere na ndị nwere ahụike yana sera 244 sitere na nodules benign sitere na ndị mmadụ na-eme nyocha ahụike kwa afọ na Ngalaba Nchịkwa na Mgbochi Ọrịa Cancer, Sun Yat-sen University Cancer Center, na 166 nodules benign.ọbara.A nakọtara adenocarcinomas ngụgụ nke Stage I site na Sun Yat-sen University Cancer Center.N'ime otu nkwado nke mpụga, enwere ikpe 48 nke nodules na-adịghị mma, ikpe 39 nke ogbo m iku ume adenocarcinoma sitere na Hospitallọ Ọgwụ Mbụ nke Mahadum Sun Yat-sen, na ikpe 24 nke ogbo I iku ume adenocarcinoma sitere na Zhengzhou Cancer Hospital.Sun Yat-sen University Cancer Center achịkọtakwara 16 ikpe nke ogbo I squamous cell lung cancer iji nwalee ike diagnostics nke metabolic classifier guzosie ike (e gosiri na onye ọrịa na-egosi na Mgbakwunye Table 5).Achịkọtara ihe nlele sitere na otu ndị nchọpụta na ndị otu nkwado ime n'etiti Jenụwarị 2018 na Mee 2020. Achịkọtara ihe atụ maka otu nkwado mpụga n'etiti Ọgọst 2021 na Ọktoba 2022. Iji wedata ajọ mbunobi nwoke na nwanyị, e kenyere ihe dị ka nha nha nha nke nwoke na nwanyị na nke ọ bụla. otu.Otu nchọpụta na otu nyocha nke ime.Ekpebiri nwoke na nwanyị so na ya dabere na mkpesa onwe onye.Enwetara nkwenye sitere n'aka ndị niile sonyere na enweghị nkwụghachi ụgwọ.Ndị nwere nodules benign bụ ndị nwere akara CT kwụsiri ike na afọ 2 ruo 5 n'oge nyocha, belụsọ maka ikpe 1 sitere na nlele nkwado mpụga, nke anakọtara tupu oge a wee chọpụta ya site na histopathology.Ewezuga bronchitis na-adịghị ala ala.A na-anakọta ikpe adenocarcinoma akpa ume tupu nkuku ume wee kwado ya site na nchọpụta ọrịa.A na-anakọta ihe nlele ọbara na-ebu ọnụ na tubes nkewa serum na-enweghị ọgwụ mgbochi ọ bụla.A na-achichi ihe nlele ọbara maka elekere 1 n'ime ụlọ wee kechie ya na 2851 × g maka nkeji 10 na 4 Celsius C iji nakọta serum supernatant.A na-ajụ oyi n'ahụ aliquots serum na -80 Celsius ruo mgbe mmịpụta metabolite.Ngalaba Mgbochi Ọrịa Cancer na Nnyocha ahụike nke Sun Yat-sen University Cancer Center chịkọtara ọdọ mmiri sitere na 100 ndị na-enye ahụike ahụike, gụnyere ọnụ ọgụgụ ndị ikom na ndị inyom dị afọ 40 ruo 55.A na-agwakọta mpịakọta nha nhata nke ihe nlele onye ọ bụla na-enye onyinye, a na-ekpochapụ ọdọ mmiri ahụ ma chekwaa ya na -80 Celsius.A na-eji ngwakọta serum mee ihe dị ka ihe ntụaka maka njikwa mma na nhazi data.
A na-agbaze serum na ihe nlele ule wee wepụta metabolites site na iji usoro mmịpụta jikọtara ọnụ (MTBE/methanol/water) 56.Na nkenke, 50 μl nke serum jikọtara ya na 225 μl nke methanol oyi na 750 μl nke ice-oyi methyl tert-butyl ether (MTBE).Gwakọta ngwakọta ma tinye na ice maka 1 hour.A na-agwakọta ihe atụ ndị ahụ na vortex jikọtara ya na 188 μl nke mmiri MS-grade nwere ụkpụrụ ime (13C-lactate, 13C3-pyruvate, 13C-methionine, na 13C6-isoleucine, nke zụtara n'aka Cambridge Isotope Laboratories).Mgbe ahụ, a na-agbanye ngwakọta ahụ na 15,000 × g maka 10 min na 4 Celsius C, a na-ebufe akụkụ nke ala n'ime tubes abụọ (125 μL ọ bụla) maka nyocha LC-MS na ụdị dị mma na nke na-adịghị mma.N'ikpeazụ, a na-ekpochapụ ihe nlele ahụ ka ọ bụrụ nkụ n'ime ngwa ngwa ngwa ngwa ngwa ngwa.
A na-emegharị metabolites a mịrị amị na 120 μl nke 80% acetonitrile, vortexed maka 5 min, na centrifuged na 15,000 × g maka 10 min na 4 ° C.Ebufere ihe ndị dị elu n'ime vial enyo amber nwere ihe ntinye microinsert maka ọmụmụ metabolomics.Nyocha metabolomics na-enweghị ebumnuche na ikpo okwu chromatography mmiri na-arụ ọrụ nke ọma-elu-mkpebi mass spectrometry (UPLC-HRMS).Ekewara metabolites site na iji usoro Dionex Ultimate 3000 UPLC yana kọlụm ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Waters).Na ọnọdụ ion ziri ezi, usoro mkpanaka bụ 95% (A) na 50% acetonitrile (B), nke ọ bụla nwere 10 mmol/L ammonium acetate na 0.1% formic acid.Na ọnọdụ na-adịghị mma, usoro mkpanaka A na B nwere 95% na 50% acetonitrile, n'otu n'otu, akụkụ abụọ ahụ nwere 10 mmol / L ammonium acetate, pH = 9. Usoro gradient dị ka ndị a: 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. A na-edobe kọlụm na 40 Celsius C na ihe nlele na 10 ° C na autosampler.Ọnụego ya bụ 0.3 ml / min, olu injection bụ 3 μl.A na-arụ ọrụ spectrometer Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) nwere isi iyi electrospray ionization (ESI) na ọnọdụ nyocha zuru oke yana yana ddMS2 nlekota ọnọdụ iji nakọta nnukwu data.Edere paramita MS dị ka ndị a: ịgbasa voltaji + 3.8 kV/- 3.2 kV, capillary okpomọkụ 320°C, mkpuchi gas 40 arb, inyeaka gas 10 arb, nyocha okpomọkụ okpomọkụ 350°C, nyocha nso 70-1050 m / h, mkpebi.70 000. E nwetara data site na iji Xcalibur 4.1 (Thermo Fisher Scientific).
Iji nyochaa ogo data, a na-emepụta ihe nlele njikwa mma (QC) site na iwepụ 10 μL aliquots nke supernatant na nlele ọ bụla.A tụlere ntụtụ ntụle njikwa mma isii na mmalite nke usoro nyocha iji chọpụta nkwụsi ike nke sistemu UPLC-MS.A na-ewebata ihe nlele njikwa ogo n'ime oge.LC-MS tụlere batches 11 niile nke ihe nlele serum na ọmụmụ a.A na-eji Aliquots nke ngwakọta ọdọ mmiri serum sitere na 100 ndị nyere ahụike dị mma dị ka ihe ntụnye aka na batches dị iche iche iji nyochaa usoro mmịpụta wee gbanwee maka mmetụta batch-to-batch.A na-eme nyocha metabolomics na-enweghị ebumnuche nke otu nchọta, otu nkwado ime, na otu nkwado mpụga na Metabolomics Center nke Mahadum Sun Yat-sen.Ụlọ nyocha dịpụrụ adịpụ nke Mahadum Guangdong nke Nkà na ụzụ Nyocha na Ụlọ Nnwale nyochakwara ihe nlele 40 sitere na otu ndị ọzọ iji nwalee arụmọrụ nke ụdị nhazi ọkwa.
Mgbe mmịpụta na mweghachi, a tụrụ ọnụọgụ zuru oke nke metabolites serum site na iji ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) na isi iyi electrospray ionization (ESI) na otutu mmeghachi omume nlekota oru (MMRM).A na-eji kọlụm ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Mmiri) kewaa metabolites.Usoro mkpanaka nwere 90% (A) na 5% acetonitrile (B) nwere 10 mmol/L ammonium acetate na 0.1% amonia ngwọta.Mmemme gradient bụ otu a: 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.A na-edobe kọlụm na 40 Celsius C na ihe nlele na 10 Celsius C na autosampler.Onu ogugu bu 0.3 ml/min na olu ogwu ogwu bu 1 μL.Edere paramita MS dị ka ndị a: voltaji capillary ± 3.5 kV, nebulizer mgbali 35 psi, n'ọbọ gas eruba 12 L / min, n'ọbọ gas okpomọkụ 350 ° C, ihicha gas okpomọkụ 250 ° C, na ihicha gas eruba 14 l / min.Ntughari MRM nke tryptophan, pyruvate, lactate, hypoxanthine na xanthine bụ 205.0-187.9, 87.0-43.4, 89.0-43.3, 135.0-92.3 na 151.0-107.9 n'otu n'otu.Ewekọtara data site na iji Mass Hunter B.07.00 (Agilent Technologies).Maka nlele ọbara, tryptophan, pyruvate, lactate, hypoxanthine na xanthine ka a na-agbakọ site na iji usoro nhazi nke ngwọta ngwakọta ọkọlọtọ.Maka nlele cell, ọdịnaya tryptophan na-ahazi ka ọ bụrụ ọkọlọtọ dị n'ime yana protein protein cell.
Emere mmịpụta kacha elu (m/z na oge njide (RT)) site na iji Compound Discovery 3.1 na TraceFinder 4.0 (Thermo Fisher Scientific).Iji kpochapụ ndịiche nwere ike ịdị n'etiti batches, e kewara ọnụ ọgụgụ kacha elu njirimara nke ihe nlele ahụ site na njirimara kacha elu nke ihe nrụtụ aka n'otu ogbe ahụ iji nweta oke nha.A na-egosi ọdịiche ọkọlọtọ nke ụkpụrụ dị n'ime n'ihu na mgbe nhazichara na tebụl mgbakwunye 6. Ọdịiche dị n'etiti otu abụọ ahụ bụ ọnụọgụ nchọpụta ụgha (FDR <0.05, Wilcoxon bịanyere aka na ọkwa ọkwa) na mpịachi mgbanwe (> 1.2 ma ọ bụ <0.83).A na-egosi data Raw MS nke njirimara ewepụtara yana data MS emeziri serum na Mgbakwunye Data 1 na Data 2, n'otu n'otu.Emere nkọwa kachasị elu dabere na ọkwa njirimara anọ akọwapụtara, gụnyere metabolites achọpụtara, ogige etinyere nke etinyere, klas ndị a na-etinye n'ụdị etinyere, na ogige amaghị ama 22.Dabere na nchọta nchekwa data dị na Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), ogige ndu nwere MS/MS dakọtara ụkpụrụ akwadoro ma ọ bụ nkọwa egwuregwu ziri ezi na mzCloud (akara> 85) ma ọ bụ Chemspider mechara họrọ dị ka ndị etiti n'etiti metabolome dị iche.Agụnyere nkọwa kacha elu maka njirimara nke ọ bụla na data mgbakwunye 3. MetaboAnalyst 5.0 ka ejiri mee nyocha otu nke nchikota metabolite nchikota.MetaboAnalyst 5.0 tụlekwara nyocha nkwalite ụzọ KEGG dabere na metabolites dị iche iche.A tụlere nyocha nke isi akụrụngwa (PCA) na nyocha ịkpa oke n'akụkụ akụkụ pere mpe (PLS-DA) site na iji ngwugwu sọftụwia ropls (v.1.26.4) na nchikota nchikota na autoscaling.Ihe nlere biomarker metabolite kacha mma maka ibu amụma mmebi nodule ka emepụtara site na iji ọnụọgụ abụọ logistic regression nwere obere mbelata na onye ọrụ nhọrọ (LASSO, R ngwugwu v.4.1-3).Arụ ọrụ nke ụdị ịkpa ókè na nchọta na nhazi nkwado bụ nke e ji atụle AUC dabere na nyocha ROC dịka ngwugwu pROC (v.1.18.0.).Enwetara mbelata ihe puru omume kacha mma dabere na ndeksi Youden kachasị nke ihe nlereanya (uche + nkọwa - 1).A ga-ebu amụma ihe atụ ndị nwere ụkpụrụ na-erughị ma ọ bụ karịa nke ọnụ ụzọ dị ka nodules na-adịghị mma na adenocarcinoma ngụgụ, n'otu n'otu.
A549 mkpụrụ ndụ (#CCL-185, American Ụdị Culture mkpokọta) ka eto na F-12K ọkara nwere 10% FBS.Ụdị ntutu dị mkpụmkpụ RNA (shRNA) nke na-elekwasị anya SLC7A5 na njikwa na-adịghị mkpa (NC) ka etinyere na vector lentiviral pLKO.1-puro.Usoro antisense nke shSLC7A5 bụ ndị a: Sh1 (5′-GGAGAAACCTGATGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGAACTAT-3′).Azụtara ọgwụ mgbochi SLC7A5 (#5347) na tubulin (#2148) site na teknụzụ nrịbama cell.A na-eji ọgwụ mgbochi na SLC7A5 na tubulin mee ihe na mgbaze nke 1:1000 maka nyocha Western blot.
The Seahorse XF Glycolytic Stress Test na-atụle ọkwa extracellular acidification (ECAR).Na nyocha, glucose, oligomycin A, na 2-DG ka a na-enye usoro iji nwalee ikike glycolytic cell dị ka ECAR tụrụ.
A na-atụgharị mkpụrụ ndụ A549 site na njikwa na-enweghị ebumnuche (NC) na shSLC7A5 (Sh1, Sh2) n'otu abalị na efere 10 cm n'obosara.A na-ewepụta metabolites cell site na 1 ml nke mmiri oyi 80% methanol.A na-ekpochapụ mkpụrụ ndụ ndị dị na ngwọta methanol, chịkọta ya na tube ọhụrụ, na centrifud na 15,000 × g maka 15 min na 4 ° C.Chịkọta 800 µl nke ihe dị elu wee kpoo ya site na iji ihe mgbanye oghere dị elu.A na-enyocha pellets metabolite akpọnwụwo maka ọkwa tryptophan site na iji LC-MS/MS dịka akọwara n'elu.A tụrụ ọkwa NAD (H) cellular na sel A549 (NC na shSLC7A5) site na iji ngwa NAD +/NADH colorimetric quantitative (#K337, BioVision) dịka ntuziaka onye nrụpụta siri dị.A tụrụ ọkwa protein maka nlele ọ bụla iji mee ka ọnụọgụ metabolites dị mma.
Ejighị ụzọ ndekọ ọnụ ọgụgụ buru ụzọ chọpụta nha nlele ahụ.A na-ahụta ọmụmụ metabolomics gara aga nke chọrọ nchọpụta biomarker15,18 dị ka akara maka ikpebi nha, ma atụnyere akụkọ ndị a, nlele anyị zuru oke.Ọnweghị ihe atụ ewepụrụ na otu ọmụmụ.E kenyere ihe nlele ọbara n'otu ntabi anya na otu nchọpụta (ikpe 306, 74.6%) yana otu nkwado dị n'ime (ikpe 104, 25.4%) maka ọmụmụ metabolomics na-atụghị anya ya.Anyị na-ahọrọkwa ikpe iri asaa n'otu n'otu na-enweghị usoro site na nchọpụta ederede maka ọmụmụ metabolomics ezubere iche.Ndị nchọpụta ahụ kpuru ìsì maka ọrụ otu n'oge nchịkọta na nyocha data LC-MS.A na-akọwa nyocha ndekọ ọnụ ọgụgụ nke data metabolomics na nnwale cell n'ime nso nso a, akụkọ ifo, na usoro.A na-eme ọnụọgụ tryptophan cellular, NADT na glycolytic ugboro atọ n'adabereghị na nsonaazụ yiri ya.
Maka ozi ndị ọzọ gbasara nhazi ọmụmụ, lee akwụkwọ akụkọ akụkọ Pọtụfoliyo eke jikọtara ya na isiokwu a.
A na-egosi data raw MS nke njirimara ewepụtara yana data MS nke ahaziri ahazi nke serum ntụaka na Data Mgbakwụnye 1 na data mgbakwunye 2, n'otu n'otu.E gosipụtara nkọwa kacha elu maka njirimara dị iche iche na Data Mmekọ 3. Enwere ike ibudata ihe ndekọ data LUAD TCGA na https://portal.gdc.cancer.gov/.A na-enye data ntinye maka nhazi eserese ahụ na data isi mmalite.Enyere data isi mmalite maka akụkọ a.
Otu ọmụmụ nyocha akpa ume mba, wdg. Mbelata ọnwụ ọrịa kansa akpa ume site na iji tomography gbakọọ obere dose.Northern England.J. Med.365, 395–409 (2011).
Kramer, BS, Berg, KD, Aberle, DR na onye amụma, PC nyocha ọrịa cancer akpa ume na-eji obere CT helical : nsonaazụ sitere na Nnyocha nyocha nyocha nke mba (NLST).J. Med.Ihuenyo 18, 109-111 (2011).
De Koning, HJ, et al.Mbelata ọnwụ cancer akpa ume site na nyocha volumetric CT na nnwale enweghị usoro.Northern England.J. Med.382, 503–513 (2020).
Oge nzipu: Sep-18-2023