Nchọpụta dị iche iche nke nodules pulmonary nke ejiri kọmputa kọwaa (CT) ka bụ ihe ịma aka n'ọrụ ahụike. N'ebe a, anyị na-akọwa metabolome zuru ụwa ọnụ nke ihe nlele serum 480, gụnyere njikwa ahụike, nodules benign ngụgụ, na adenocarcinoma akpa ume nke ọkwa nke mbụ. Adenocarcinomas na-egosipụta profaịlụ metabolomik pụrụ iche, ebe nodules benign na ndị nwere ahụike nwere nnukwu myirịta na profaịlụ metabolomik. Na otu nchọpụta (n = 306), achọpụtara otu metabolites 27 iji mata ọdịiche dị n'etiti nodules benign na nke dị njọ. AUC nke ụdị dị iche iche na nkwenye ime (n = 104) na otu nkwenye mpụga (n = 111) bụ 0.915 na 0.945, n'otu n'otu. Nyocha ụzọ gosiri mmụba nke metabolites glycolytic jikọtara ya na mbelata tryptophan na serum adenocarcinoma akpa ume ma e jiri ya tụnyere nodules benign na njikwa ahụike, ma tụọ aro na nnabata tryptophan na-akwalite glycolysis na mkpụrụ ndụ kansa akpa ume. Ọmụmụ ihe anyị na-egosi uru nke ihe ndị na-egosi metabolite serum n'inyocha ihe egwu nke nodules akpa ume nke CT chọpụtara.
Nchọpụta mbụ dị oke mkpa iji melite ọnụọgụ ndụ nke ndị ọrịa kansa. Nsonaazụ sitere na nnwale nyocha ọrịa kansa akpa ume nke mba US (NLST) na ọmụmụ ihe NELSON nke Europe egosila na inyocha site na iji obere dose nke kọmputa tomography (LDCT) nwere ike ibelata ọnwụ nke kansa akpa ume n'ime otu ndị nwere nnukwu ihe egwu1,2,3. Kemgbe ojiji LDCT n'ọtụtụ ebe maka nyocha kansa akpa ume, ọnụọgụ nke nchọpụta rediographic mberede nke nodules pulmonary asymptomatic anọgidewo na-abawanye 4. A na-akọwa nodules pulmonary dị ka opacity focal ruo 3 cm na dayameta 5. Anyị na-eche nsogbu ihu n'inyocha ohere nke ọrịa kansa na ịnagide ọnụ ọgụgụ buru ibu nke nodules pulmonary achọpụtara na mberede na LDCT. Mmachi nke CT nwere ike ibute nyocha ugboro ugboro na nsonaazụ ụgha, na-eduga na ntinye aka na-enweghị isi na ọgwụgwọ karịrị akarị6. Ya mere, ọ dị mkpa ịmepụta ihe ngosi biomarkers a pụrụ ịtụkwasị obi na bara uru iji chọpụta ọrịa kansa akpa ume nke ọma na mbido ma chọpụta ọtụtụ nodules na-adịghị mma na nchọpụta mbụ 7.
Nnyocha zuru oke nke ọbara (serum, plasma, mkpụrụ ndụ mononuclear ọbara dị n'akụkụ), gụnyere genomics, proteomics ma ọ bụ DNA methylation8,9,10, emeela ka mmasị na-eto eto na nchọpụta nke biomarkers nchọpụta maka ọrịa kansa akpa ume. Ka ọ dị ugbu a, ụzọ metabolomics na-atụle ngwaahịa njedebe sel nke ihe omume endogenous na exogenous na-emetụta ma ya mere a na-etinye ya iji buo amụma mmalite na nsonaazụ ọrịa. Liquid chromatography-tandem mass spectrometry (LC-MS) bụ usoro a na-ejikarị eme ihe maka ọmụmụ ihe metabolomics n'ihi nnukwu mmetụta ya na oke ike dị iche iche, nke nwere ike ikpuchi metabolites nwere ihe dị iche iche nke physicochemical11,12,13. Ọ bụ ezie na ejirila nyocha metabolomik zuru ụwa ọnụ nke plasma/serum chọpụta biomarkers metụtara nchọpụta kansa akpa ume14,15,16,17 na irè ọgwụgwọ, a ka ga-amụ ọtụtụ ihe gbasara klaasị metabolite serum 18 iji mata ọdịiche dị n'etiti nodules akpa ume dị mma na nke na-adịghị mma. - nnukwu nnyocha.
Ọrịa kansa nke Adenocarcinoma na squamous cell carcinoma bụ ụdị abụọ bụ isi nke ọrịa kansa akpa ume na-abụghị obere sel (NSCLC). Nnwale CT dị iche iche na-egosi na adenocarcinoma bụ ụdị kansa akpa ume kachasị na histological1,19,20,21. N'ọmụmụ ihe a, anyị jiri ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) mee nyocha metabolomics na mkpokọta ihe nlele serum 695, gụnyere njikwa ahụike, benign pulmonary nodules, na CT-achọpụtara ≤3 cm. Nyocha maka adenocarcinoma akpa ume nke Stage I. Anyị chọpụtara otu panel nke serum metabolites nke na-amata adenocarcinoma akpa ume site na benign nodules na njikwa ahụike. Nyocha mmụba ụzọ kpughere na tryptophan na-adịghị mma na metabolism glucose bụ mgbanwe a na-ahụkarị na adenocarcinoma akpa ume ma e jiri ya tụnyere benign nodules na njikwa ahụike. N'ikpeazụ, anyị guzobere ma kwado usoro nhazi metabolic serum nke nwere nkọwapụta na mmetụta dị elu iji mata ọdịiche dị n'etiti nodules pulmonary dị njọ na nke na-adịghị mma nke LDCT chọpụtara, nke nwere ike inye aka na nchọpụta ọdịiche dị n'oge na nyocha ihe egwu.
N'ọmụmụ ihe a, e si n'aka mmadụ 174 nwere ahụike, ndị ọrịa 292 nwere ọrịa akpa ume dị mma, na ndị ọrịa 229 nwere ọrịa akpa ume nke ọkwa nke mbụ nweta ihe nlele ọbara ọbara. E gosiri njirimara mmadụ nke ndị ọrịa 695 na Tebụl Mgbakwunye 1.
Dịka egosiri na Foto 1a, a chịkọtara ihe nlele serum 480, gụnyere ihe nlele ahụike 174 (HC), nodules 170 benign (BN), na ihe nlele 136 nke akpa ume adenocarcinoma (LA), na Sun Yat-sen University Cancer Center. Ndị otu Discovery maka profaịlụ metabolic na-enweghị ebumnuche site na iji ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Dịka egosiri na Foto Mgbakwunye 1, achọpụtara metabolites dị iche n'etiti LA na HC, LA na BN iji guzobe ụdị nhazi ma nyochaa nyocha ụzọ dị iche iche. Ihe nlele 104 nke Sun Yat-sen University Cancer Center chịkọtara na ihe nlele 111 nke ụlọ ọgwụ abụọ ndị ọzọ chịkọtara ka enyere nkwenye dị n'ime na mpụga, n'otu n'otu.
a Ọnụọgụgụ ndị mmadụ nọ n'ìgwè nchọpụta nke mere nyocha metabolomics zuru ụwa ọnụ nke serum site na iji ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). b Nyocha nkewa nke akụkụ pere mpe (PLS-DA) nke mkpokọta metabolome nke ihe nlele serum 480 sitere na otu ọmụmụ ihe ahụ, gụnyere njikwa ahụike (HC, n = 174), nodules na-adịghị mma (BN, n = 170), na adenocarcinoma akpa ume nke ọkwa I (Los Angeles, n = 136). +ESI, ọnọdụ ionization electrospray dị mma, -ESI, ụdị ionization electrospray na-adịghị mma. c–e Metabolites nwere ọtụtụ ihe dị iche iche na otu abụọ enyere (ule ọkwa Wilcoxon nke nwere ọdụ abụọ, ọnụego nchọpụta ụgha agbanwere uru p, FDR <0.05) ka egosiri na uhie (mgbanwe mpịachi > 1.2) na acha anụnụ anụnụ (mpịachi mpịachi < 0.83). ) egosiri na eserese ugwu mgbawa ahụ. f Maapụ okpomọkụ nke nhazi ọkwa na-egosi nnukwu ọdịiche dị na ọnụọgụ nke metabolites edepụtara n'etiti LA na BN. A na-enye data isi mmalite n'ụdị faịlụ data isi mmalite.
E jiri nyocha UPLC-HRMS nyochaa mkpokọta metabolome nke 174 HC, 170 BN na 136 LA n'ime otu nchọpụta ahụ. Anyị buru ụzọ gosi na ihe nlele njikwa mma (QC) na-agbakọta nke ọma n'etiti ihe nlereanya nyocha akụkụ isi (PCA) nke a na-anaghị elekọta, na-akwado nkwụsi ike nke arụmọrụ ọmụmụ ihe a ugbu a (Foto mgbakwunye 2).
Dịka egosiri na nyocha nke obere akụkụ anọ (PLS-DA) na Foto 1 b, anyị chọpụtara na e nwere ọdịiche doro anya n'etiti LA na BN, LA na HC na ụdị ionization electrospray dị mma (+ESI) na nke na-adịghị mma (−ESI). e kewapụrụ. Agbanyeghị, a hụghị ọdịiche dị mkpa n'etiti BN na HC na ọnọdụ +ESI na -ESI.
Anyị hụrụ atụmatụ dị iche iche 382 dị n'etiti LA na HC, atụmatụ dị iche iche 231 dị n'etiti LA na BN, na atụmatụ dị iche iche 95 dị n'etiti BN na HC (ule ọkwa Wilcoxon bịanyere aka na ya, FDR <0.05 na mgbanwe dị iche iche >1.2 ma ọ bụ <0.83) (Foto .1c-e). . E dekọrọ ihe ndị dị elu (Data Mgbakwunye 3) megide nchekwa data (ọbá akwụkwọ mzCloud/HMDB/Chemspider) site na uru m/z, oge njide na nchọta oke ekewa (nkọwa akọwapụtara na ngalaba Usoro) 22. N'ikpeazụ, achọpụtara metabolites 33 na 38 nwere nnukwu ọdịiche dị n'ọtụtụ maka LA megide BN (Foto 1f na Tebụl Mgbakwunye 2) na LA megide HC (Foto Mgbakwunye 3 na Tebụl Mgbakwunye 2), n'otu n'otu. N'ụzọ dị iche, naanị metabolites atọ nwere nnukwu ọdịiche dị n'ọtụtụ ka achọpụtara na BN na HC (Tebụl Mgbakwunye 2), nke kwekọrọ na njikọ dị n'etiti BN na HC na PLS-DA. Metabolites ndị a dị iche iche na-ekpuchi ọtụtụ biochemicals (Foto Mgbakwunye 4). A na-ejikọta ha ọnụ, nsonaazụ ndị a na-egosi mgbanwe dị ukwuu na metabolome serum nke na-egosipụta mgbanwe ọjọọ nke kansa akpa ume na mbido ma e jiri ya tụnyere nodules akpa ume na-adịghị mma ma ọ bụ ndị nwere ahụike. Ka ọ dị ugbu a, myirịta nke metabolome serum nke BN na HC na-egosi na nodules pulmonary na-adịghị mma nwere ike ịkekọrịta ọtụtụ njirimara bayoloji na ndị nwere ahụike. Ebe ọ bụ na mgbanwe mkpụrụ ndụ ihe nketa epidermal growth factor receptor (EGFR) bụ ihe a na-ahụkarị na adenocarcinoma akpa ume subtype 23, anyị gbalịrị ịchọpụta mmetụta nke mgbanwe ndị ọkwọ ụgbọala na metabolome serum. Anyị nyochachara profaịlụ metabolomik zuru oke nke ikpe 72 nwere ọnọdụ EGFR na otu adenocarcinoma akpa ume. N'ụzọ na-akpali mmasị, anyị hụrụ profaịlụ yiri nke ahụ n'etiti ndị ọrịa nwere mgbanwe EGFR (n = 41) na ndị ọrịa nwere ụdị anụ ọhịa EGFR (n = 31) na nyocha PCA (Foto mgbakwunye 5a). Agbanyeghị, anyị chọpụtara metabolites 7 nke ọnụọgụ ha gbanwere nke ukwuu na ndị ọrịa nwere mgbanwe EGFR ma e jiri ya tụnyere ndị ọrịa nwere ụdị anụ ọhịa EGFR (nnwale t, p < 0.05 na mgbanwe mpịachi > 1.2 ma ọ bụ < 0.83) (Foto mgbakwunye 5b). Ọtụtụ n'ime metabolites ndị a (5 n'ime 7) bụ acylcarnitines, nke na-arụ ọrụ dị mkpa na ụzọ oxidation acid fatty.
Dịka egosiri na usoro ọrụ egosiri na Foto 2 a, e nwetara ihe nrịbama maka nhazi nodule site na iji ndị na-arụ ọrụ mbelata ihe na nhọrọ kachasị nta dabere na metabolites dị iche iche 33 achọpụtara na LA (n = 136) na BN (n = 170). Nchikota kachasị mma nke mgbanwe (LASSO) - ụdị mgbanwe logistic binary. Ejiri okpukpu iri nke nkwenye cross-validation nwalee ntụkwasị obi nke ihe nlereanya ahụ. A na-agbanwe nhọrọ mgbanwe na nhazi paramita site na ntaramahụhụ kachasị mma nke nwere paramita λ24. Emere nyocha metabolomics zuru ụwa ọnụ n'adabereghị onwe ya na otu nkwenye dị n'ime (n = 104) na nkwenye mpụga (n = 111) iji nwalee arụmọrụ nhazi nke ihe nlereanya nkewa. N'ihi ya, achọpụtara metabolites 27 na setịpụ nchọpụta dị ka ihe nlereanya kacha mma nkewa nwere uru AUC kachasị ukwuu (Fig. 2b), n'ime ha 9 nwere mmụba ọrụ na 18 mbelata ọrụ na LA ma e jiri ya tụnyere BN (Fig. 2c).
Usoro ọrụ maka iwulite nhazi nodule pulmonary, gụnyere ịhọrọ panel kacha mma nke metabolites serum na set nchọpụta site na iji ụdị mgbanwe logistic binary site na nhazi cross-validation okpukpu iri na ịtụle arụmọrụ amụma na set nkwenye dị n'ime na mpụga. b Ọnụọgụ nkwenye obe nke ụdị regression LASSO maka nhọrọ biomarker metabolic. Ọnụọgụ enyere n'elu na-anọchite anya ọnụọgụ nke biomarker ahọpụtara na λ enyere. Ahịrị ntụpọ uhie na-anọchite anya uru AUC nkezi na lambda kwekọrọ. Ogwe njehie isi awọ na-anọchite anya uru AUC kacha nta na kachasị. Ahịrị ntụpọ na-egosi ụdị kacha mma yana biomarker 27 ahọpụtara. AUC, mpaghara n'okpuru usoro njirimara ọrụ nnata (ROC). c Mgbanwe mpịachi nke metabolites 27 ahọpụtara na otu LA ma e jiri ya tụnyere otu BN na otu nchọpụta. Kọlụm uhie - mmalite. Kọlụm acha anụnụ anụnụ bụ mbelata. d–f Usoro njirimara ọrụ nnata (ROC) na-egosi ike nke ụdị nkewa dabere na njikọta metabolite 27 na set nkwenye nchọpụta, ime, na mpụga. A na-enye data isi mmalite n'ụdị faịlụ data isi mmalite.
E mepụtara ihe nlereanya amụma dabere na ọnụọgụgụ mgbanwe dị arọ nke metabolites 27 ndị a (Tebụl Mgbakwunye 3). Nyocha ROC dabere na metabolites 27 ndị a nyere mpaghara n'okpuru uru (AUC) nke 0.933, mmetụta otu nchọpụta bụ 0.868, nkọwapụta bụ 0.859 (Fig. 2d). Ka ọ dị ugbu a, n'ime metabolites dị iche iche 38 e depụtara n'etiti LA na HC, otu metabolites 16 nwetara AUC nke 0.902 yana mmetụta nke 0.801 na nkọwapụta nke 0.856 na ịkpa ókè nke LA na HC (Foto Mgbakwunye 6a-c). A tụnyekwara ụkpụrụ AUC dabere na oke mgbanwe mpịachi dị iche iche maka metabolites dị iche iche. Anyị chọpụtara na ihe nlereanya nhazi ahụ rụrụ ọrụ nke ọma n'ịkpa ókè n'etiti LA na BN (HC) mgbe edobere ọkwa mgbanwe mpịachi na 1.2 megide 1.5 ma ọ bụ 2.0 (Foto Mgbakwunye 7a,b). A kwadoro ihe nlereanya nhazi ahụ, nke dabere na otu metabolite 27, n'ime otu ndị dị n'ime na n'èzí. AUC bụ 0.915 (sensitivity 0.867, specificity 0.811) maka nkwenye ime na 0.945 (sensitivity 0.810, specificity 0.979) maka nkwenye mpụga (Fig. 2e, f). Iji nyochaa arụmọrụ interlaboratory, a nyochachara ihe nlele 40 sitere na otu ndị dị n'èzí n'ime ụlọ nyocha mpụga dịka akọwara na ngalaba Usoro. Izi ezi nke nhazi ahụ nwetara AUC nke 0.925 (Foto mgbakwunye 8). N'ihi na ọrịa kansa squamous cell ngụgụ (LUSC) bụ ụdị nke abụọ kachasị na-ahụkarị nke kansa akpa ume na-abụghị obere mkpụrụ ndụ (NSCLC) mgbe adenocarcinoma akpa ume (LUAD), anyị nwalekwara uru nwere ike nke profaịlụ metabolic. BN na ikpe 16 nke LUSC. AUC nke ịkpa ókè n'etiti LUSC na BN bụ 0.776 (Foto mgbakwunye 9), na-egosi ike adịghị mma ma e jiri ya tụnyere ịkpa ókè n'etiti LUAD na BN.
Nnyocha egosila na nha nke nodules na onyonyo CT nwere njikọ dị mma na ohere nke ọrịa kansa ma ka bụ isi ihe na-ekpebi ọgwụgwọ nodules25,26,27. Nyocha nke data sitere na nnukwu ìgwè nke ọmụmụ nyocha NELSON gosiri na ihe egwu nke ọrịa kansa na ndị nwere nodes <5 mm yiri nke ndị na-enweghị nodes 28. Ya mere, obere nha chọrọ nlekota CT mgbe niile bụ 5 mm, dịka British Thoracic Society (BTS) kwadoro, na 6 mm, dịka Fleischner Society 29 kwadoro. Agbanyeghị, nodules buru ibu karịa 6 mm na enweghị njirimara doro anya, nke a na-akpọ indeterminate pulmonary nodules (IPN), ka bụ nnukwu ihe ịma aka na nyocha na njikwa na omume ahụike30,31. Anyị nyochachara ma nha nodules metụtara mbinye aka metabolic site na iji ihe atụ ndị a chịkọtara site na nchọpụta na ndị otu nkwenye dị n'ime. Na-elekwasị anya na biomarkers 27 akwadoro, anyị buru ụzọ tụnyere profaịlụ PCA nke HC na BN sub-6 mm metabolomes. Anyị chọpụtara na ọtụtụ ebe data maka HC na BN jikọtara ọnụ, na-egosi na ọkwa metabolite serum yiri nke ahụ n'ìgwè abụọ ahụ (Foto 3a). Maapụ atụmatụ ndị dị n'ofe nha dị iche iche ka echekwara na BN na LA (Foto 3b, c), ebe a hụrụ nkewa n'etiti nodules ọjọọ na nke na-adịghị mma na oke 6-20 mm (Foto 3d). Ndị otu a nwere AUC nke 0.927, nkọwapụta nke 0.868, na mmetụta nke 0.820 maka ịkọ amụma ihe ọjọọ nke nodules nke ruru 6 ruo 20 mm (Foto 3e, f). Nsonaazụ anyị na-egosi na ihe nhazi ahụ nwere ike ijide mgbanwe metabolic nke mgbanwe kansa mbụ kpatara, n'agbanyeghị nha nodule.
Ntụnyere nke profaịlụ PCA n'etiti otu akọwapụtara dabere na nhazi 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 Usoro njirimara ọrụ nnata (ROC) na-egosi arụmọrụ ụdị dị iche iche maka nodules 6–20 mm. f A gbakọrọ ụkpụrụ ihe omume dabere na ụdị nhazi logistic maka nodules ndị dị nha 6–20 mm. Ahịrị ntụpọ isi awọ na-anọchite anya uru mbepụ kacha mma (0.455). Ọnụọgụ ndị dị n'elu na-anọchite anya pasent nke ikpe ndị a tụrụ anya maka Los Angeles. Jiri ule t nke ụmụ akwụkwọ nwere ọdụ abụọ. PCA, nyocha akụkụ bụ isi. Mpaghara AUC n'okpuru usoro. A na-enye data isi mmalite n'ụdị faịlụ data isi mmalite.
A họrọkwara ihe nlele anọ (ndị dị afọ 44–61) nwere nha ngụgụ akpa ume yiri nke ahụ (7–9 mm) iji gosi arụmọrụ nke ihe nlereanya amụma ọrịa kansa akwadoro (Foto 4a, b). Na nyocha mbụ, a gosipụtara Ikpe 1 dị ka nodule siri ike nwere calcification, njirimara metụtara benignity, ebe Ikpe 2 gosipụtara dị ka nodule siri ike nke na-enweghị njirimara doro anya. Agba atọ nke nyocha CT na-esochi gosiri na ikpe ndị a nọgidere na-eguzosi ike n'ihe ruo afọ 4 ma ya mere a na-ewere ha dị ka nodules na-adịghị mma (Foto 4a). Ma e jiri ya tụnyere nyocha ahụike nke nyocha CT usoro, nyocha metabolite serum otu-ose na ụdị nhazi ugbu a ngwa ngwa ma chọpụta nodules ndị a na-adịghị mma nke ọma dabere na mgbochi puru omume (Tebụl 1). Foto 4b na ikpe 3 na-egosi nodule nwere ihe ịrịba ama nke mweghachi pleural, nke a na-ejikọkarị ya na malignancy32. Ikpe 4 gosipụtara dị ka nodule siri ike nke na-enweghị ihe akaebe nke ihe kpatara ya. E buru amụma na ikpe ndị a niile ga-abụ ihe ọjọọ dịka ihe nlereanya nkewa (Tebụl 1). E gosipụtara nyocha nke adenocarcinoma akpa ume site na nyocha histopathological mgbe a wachara ya ahụ (Foto 4b). Maka nhazi nkwenye mpụga, nkewa metabolic buru amụma nke ọma ikpe abụọ nke nodules akpa ume na-enweghị nkọwa nke buru ibu karịa 6 mm (Foto mgbakwunye 10).
Foto CT nke windo axial nke akpa ume nke ikpe abụọ nke nodules na-adịghị mma. N'ọnọdụ 1, nyocha CT mgbe afọ 4 gasịrị gosiri nodule siri ike kwụsiri ike nke ruru 7 mm yana calcification na lobe aka nri ala. N'ọnọdụ 2, nyocha CT mgbe afọ 5 gasịrị gosiri nodule kwụsiri ike, nke siri ike nke nwere dayameta nke 7 mm na lobe aka nri elu. b Foto CT windo axial nke akpa ume na ọmụmụ ihe gbasara ọrịa nke ikpe abụọ nke adenocarcinoma nke ọkwa I tupu mwepụ akpa ume. Ikpe nke 3 kpughere nodule nwere dayameta nke 8 mm na lobe aka nri elu yana mwepụ pleural. Ikpe nke 4 kpughere nodule ala siri ike nke dị 9 mm na lobe aka ekpe elu. Hematoxylin na eosin (H&E) agba nke anụ ahụ akpa ume a gbapụrụ agbapụ (ogwe nha = 50 μm) na-egosi usoro uto acinar nke adenocarcinoma akpa ume. Akụ na-egosi nodules achọpụtara na onyonyo CT. Foto H&E bụ onyonyo nnọchiteanya nke ọtụtụ (>3) obere ubi microscopic nke onye na-ahụ maka ọrịa nyochara.
N'ịchịkọta ihe anyị chọpụtara, ihe ndị a na-egosi uru dị na biomarkers nke serum metabolite na nchọpụta dị iche iche nke nodules pulmonary, nke nwere ike ibute nsogbu mgbe a na-enyocha nyocha CT.
Dabere na panel metabolite dị iche iche akwadoro, anyị chọrọ ịchọpụta njikọ dị n'etiti mgbanwe metabolic dị mkpa. Nyocha mmụba ụzọ KEGG site na MetaboAnalyst chọpụtara ụzọ isii a na-ahụkarị nke gbanwere nke ukwuu n'etiti otu abụọ enyere (LA vs. HC na LA vs. BN, agbanwere p ≤ 0.001, mmetụta > 0.01). A na-akọwa mgbanwe ndị a site na nsogbu na metabolism pyruvate, metabolism tryptophan, metabolism niacin na nicotinamide, glycolysis, okirikiri TCA, na metabolism purine (Fig. 5a). Anyị mekwara metabolomics ezubere iche iji chọpụta nnukwu mgbanwe site na iji quantification zuru oke. Mkpebi nke metabolites nkịtị na ụzọ ndị a na-agbanwekarị site na triple quadrupole mass spectrometry (QQQ) site na iji ezigbo ụkpụrụ metabolite. Agụnyere njirimara mmadụ nke ihe atụ ebumnuche nke ọmụmụ metabolomics na Tebụl Mgbakwunye 4. Dịka nsonaazụ metabolomics zuru ụwa ọnụ anyị si dị, nyocha ọnụọgụgụ 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, achọpụtaghị ọdịiche dị mkpa na metabolites ndị a n'etiti BN na HC.
Nnyocha mmụba ụzọ KEGG nke metabolites dị iche iche dị na otu LA ma e jiri ya tụnyere otu BN na HC. Ejiri Globaltest nwere ọdụ abụọ, e mekwara ụkpụrụ p site na iji usoro Holm-Bonferroni (agbanwere p ≤ 0.001 na nha mmetụta > 0.01). b–d violin plots na-egosi ọkwa hypoxanthine, xanthine, lactate, pyruvate, na tryptophan na serum HC, BN, na LA nke LC-MS/MS kpebiri (n = 70 kwa otu). Ahịrị ntụpọ ọcha na oji na-egosi etiti na quartile, n'otu n'otu. e violin plot na-egosi Log2TPM (ndepụta kwa nde) mRNA nke SLC7A5 na QPRT nke a na-ahazi na adenocarcinoma akpa ume (n = 513) ma e jiri ya tụnyere anụ ahụ akpa ume nkịtị (n = 59) na dataset LUAD-TCGA. Igbe ọcha ahụ na-anọchite anya oke interquartile, ahịrị ojii kwụ ọtọ dị n'etiti na-anọchite anya etiti, ahịrị ojii kwụ ọtọ nke si na igbe ahụ na-anọchite anya oge ntụkwasị obi 95% (CI). f Pearson strate nke ngosipụta SLC7A5 na GAPDH na adenocarcinoma akpa ume (n = 513) na anụ ahụ akpa ume nkịtị (n = 59) na dataset TCGA. Mpaghara isi awọ na-anọchite anya 95% CI. r, Pearson correlation coefficient. g Ọkwa tryptophan cellular nkịtị na mkpụrụ ndụ A549 nke ejiri njikwa shRNA na-abụghị nke akọwapụtara (NC) na shSLC7A5 (Sh1, Sh2) nke LC-MS/MS kpebiri. E gosipụtara nyocha ọnụọgụgụ nke ihe nlele ise nwere onwe ha na bayoloji n'otu ọ bụla. h Ọkwa sel nke NADt (ngụkọta NAD+ na NADH) na mkpụrụ ndụ A549 (NC) na SLC7A5 knockdown mkpụrụ ndụ A549 (Sh1, Sh2). E gosipụtara nyocha ọnụọgụgụ nke ihe nlele atọ nwere onwe ha na bayoloji n'otu ọ bụla. i A tụrụ ọrụ Glycolytic nke mkpụrụ ndụ A549 tupu na mgbe SLC7A5 gwụchara site na ọnụego acidification extracellular (ECAR) (n = ihe nlele anọ nke onwe ha na bayọlọji kwa otu). 2-DG,2-deoxy-D-glucose. Ejiri ule t nke ụmụ akwụkwọ nwere ọdụ abụọ mee ihe na (b–h). Na (g–i), ogwe njehie na-anọchite anya nkezi ± SD, emere nnwale ọ bụla ugboro atọ n'otu n'otu, nsonaazụ ya yikwara otu. A na-enye data isi mmalite n'ụdị faịlụ data isi mmalite.
N'ịtụle mmetụta dị ukwuu nke mgbanwe metabolism tryptophan na otu LA, anyị nyochakwara ọkwa tryptophan serum na otu HC, BN, na LA site na iji QQQ. Anyị chọpụtara na tryptophan serum belatara na LA ma e jiri ya tụnyere HC ma ọ bụ BN (p < 0.001, Foto 5d), nke kwekọrọ na nchọpụta ndị gara aga na ọkwa tryptophan na-agagharị dị ala na ndị ọrịa nwere kansa akpa ume karịa na njikwa ahụike sitere na otu njikwa33,34,35. Ọmụmụ ihe ọzọ ejiri PET/CT tracer 11C-methyl-L-tryptophan chọpụtara na oge njigide mgbaàmà tryptophan na anụ ahụ kansa akpa ume mụbara nke ukwuu ma e jiri ya tụnyere ọnya ndị na-adịghị mma ma ọ bụ anụ ahụ nkịtị36. Anyị na-eche na mbelata na tryptophan na serum LA nwere ike igosipụta nnabata tryptophan na-arụ ọrụ site na mkpụrụ ndụ kansa akpa ume.
A makwaara na ihe ikpeazụ nke ụzọ kynurenine nke catabolism tryptophan 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 ndị gara aga lekwasịrị anya n'ọrụ nke catabolism tryptophan na nhazi ahụ ji alụso ọrịa ọgụ, anyị gbalịrị ịkọwa mmekọrịta dị n'etiti dysregulation tryptophan na ụzọ glycolytic a hụrụ na ọmụmụ ihe a. A maara na ezinụlọ 7 transporter family 5 (SLC7A5) bụ onye na-ebu tryptophan43,44,45. Quinolinic acid phosphoribosyltransferase (QPRT) bụ enzyme dị n'okpuru ụzọ kynurenine nke na-agbanwe quinolinic acid ka ọ bụrụ NAMN46. Nnyocha nke dataset LUAD TCGA kpughere na ma SLC7A5 na QPRT amụbaala nke ukwuu na anụ ahụ etuto ma e jiri ya tụnyere anụ ahụ nkịtị (Fig. 5e). A hụrụ mmụba a n'ọkwa nke mbụ na nke abụọ yana nke atọ na nke anọ nke adenocarcinoma akpa ume (Foto mgbakwunye 11), na-egosi nsogbu mbụ na metabolism tryptophan nke metụtara tumorigenesis.
Tinyere nke ahụ, dataset LUAD-TCGA gosiri njikọ dị mma n'etiti ngosipụta SLC7A5 na GAPDH mRNA na ihe nlele ndị ọrịa kansa (r = 0.45, p = 1.55E-26, Foto 5f). N'ụzọ dị iche, ahụghị njikọ dị mkpa n'etiti mbinye aka mkpụrụ ndụ ihe nketa dị otú ahụ na anụ ahụ akpa ume nkịtị (r = 0.25, p = 0.06, Foto 5f). Mbelata nke SLC7A5 (Ihe osise mgbakwunye 12) na mkpụrụ ndụ A549 belatara ọkwa tryptophan na NAD(H) nke sel (Foto 5g,h), nke butere mbelata ọrụ glycolytic dịka a tụrụ site na ọnụego acidification extracellular (ECAR) (Foto 1). 5i). Ya mere, dabere na mgbanwe metabolic na nchọpụta serum na in vitro, anyị na-eche na metabolism tryptophan nwere ike imepụta NAD+ site na ụzọ kynurenine ma rụọ ọrụ dị mkpa n'ịkwalite glycolysis na kansa akpa ume.
Nnyocha egosila na ọtụtụ nodules akpa ume na-enweghị nkọwa nke LDCT chọpụtara nwere ike ibute mkpa maka nnwale ndị ọzọ dịka PET-CT, biopsy akpa ume, na ọgwụgwọ karịrị akarị n'ihi nchọpụta ihe ọjọọ na-ezighi ezi.31 Dịka egosiri na Foto nke 6, ọmụmụ ihe anyị chọpụtara otu panel nke metabolites serum nwere uru nchọpụta nwere ike ime ka nhazi ihe egwu dịkwuo mma na njikwa nke nodules akpa ume nke CT chọpụtara na-esote.
A na-enyocha eriri akpa ume site na iji obere dose nke kọmputa (LDCT) yana atụmatụ onyonyo nke na-egosi ihe kpatara ya. Ihe na-adịghị mma ma ọ bụ ihe na-akpata ya nwere ike ibute nleta ugboro ugboro, ntinye aka na-enweghị isi, na ọgwụgwọ karịrị akarị. Ntinye nke ndị na-ahazi metabolic serum nwere uru nchọpụta nwere ike ime ka nyocha ihe egwu dịkwuo mma na njikwa nke eriri akpa ume na-esote. PET positron emission tomography.
Data sitere na nnyocha NLST nke US na nyocha NELSON nke Europe na-egosi na inyocha ndị nwere nnukwu ihe egwu site na iji obere dose nke kọmputa (LDCT) nwere ike ibelata ọnwụ nke kansa akpa ume1,3. Agbanyeghị, nyocha ihe egwu na njikwa ahụike na-esote nke ọtụtụ nodules akpa ume na-apụtaghị ìhè nke LDCT chọpụtara ka bụ ihe kacha sie ike. Isi ihe mgbaru ọsọ bụ ime ka nhazi ziri ezi nke usoro dabere na LDCT dị ugbu a ka mma site na itinye ihe nrịbama a pụrụ ịdabere na ya.
A chọpụtala ụfọdụ ihe ndị na-egosi ihe dị ndụ, dị ka metabolites ọbara, site n'ịtụnyere kansa akpa ume na njikwa ahụike15,17. N'ọmụmụ ihe a, anyị lekwasịrị anya n'itinye nyocha metabolomics serum iji mata ọdịiche dị n'etiti nodules pulmonary benign na malignant nke LDCT chọpụtara n'amaghị ama. Anyị tụnyere metabolome serum zuru ụwa ọnụ nke njikwa ahụike (HC), nodules benign lung (BN), na ihe nlele adenocarcinoma akpa ume nke ọkwa nke mbụ site na iji nyocha UPLC-HRMS. Anyị chọpụtara na HC na BN nwere profaịlụ metabolic yiri nke ahụ, ebe LA gosipụtara mgbanwe dị ukwuu ma e jiri ya tụnyere HC na BN. Anyị chọpụtara usoro metabolomites serum abụọ nke dị iche na LA na HC na BN.
Usoro nchọpụta LDCT dị ugbu a maka nodules ndị na-adịghị mma na ndị na-adịghị njọ dabere na nha, njupụta, ọdịdị na ọnụego uto nke nodules ka oge na-aga30. Ọmụmụ ihe ndị gara aga egosila na nha nodules nwere njikọ chiri anya na ohere nke kansa akpa ume. Ọbụna n'ime ndị ọrịa nwere nnukwu ihe egwu, ihe ize ndụ nke kansa na nodules <6 mm bụ <1%. Ihe ize ndụ nke kansa maka nodules ndị dị n'agbata 6 ruo 20 mm sitere na 8% ruo 64%30. Ya mere, Fleischner Society na-atụ aro ka a na-egbutu dayameta nke 6 mm maka nlekota CT oge niile. 29 Agbanyeghị, a naghị eme nyocha ihe egwu na njikwa nke nodules pulmonary indeterminated (IPN) karịrị 6 mm nke ọma 31. Nlekọta ọrịa obi a mụrụ n'oge a na-adaberekarị na ichere nchere anya yana nlekota CT ugboro ugboro.
Dabere na metabolome akwadoro, anyị gosipụtara maka oge mbụ njikọta nke mbinye aka metabolic n'etiti ndị nwere ahụike na nodules na-adịghị mma <6 mm. Myirịta nke ihe ndị dị ndụ kwekọrọ na nchọpụta CT gara aga na ihe egwu nke ọrịa kansa maka nodules na-adịghị mma <6 mm dị ala dịka maka ndị na-enweghị nodules.30 Ọ dị mkpa ịmara na nsonaazụ anyị na-egosikwa na nodules na-adịghị mma <6 mm na ≥6 mm nwere nnukwu myirịta na profaịlụ metabolic, na-egosi na nkọwa ọrụ nke ọrịa na-adịghị mma na-agbanwe agbanwe n'agbanyeghị nha nodules. Ya mere, panels nke oge a na-achọpụta ọrịa serum metabolite nwere ike inye otu nnwale dị ka nnwale iwepụ mgbe achọpụtara nodules na CT na mbụ ma nwee ike ibelata nlekota usoro. N'otu oge ahụ, otu panel nke biomarkers metabolic chọpụtara nodules na-adịghị mma ≥6 mm na nha benign ma nye amụma ziri ezi maka IPNs nke nha yiri ya na atụmatụ morphological na onyonyo CT. Nkewa metabolism serum a rụrụ ọrụ nke ọma n'ịkọ amụma ihe ọjọọ nke nodules na-adịghị mma ≥6 mm na AUC nke 0.927. N'ịchịkọta ihe anyị chọpụtara, ihe ndị a chọpụtara na-egosi na ihe pụrụ iche dị na serum nwere ike igosi mgbanwe metabolic mbụ nke etuto ahụ kpatara ma nwee uru dị ka ihe ndị na-egosi ihe egwu, n'agbanyeghị nha nodule.
Ihe pụrụ iche bụ na ọrịa kansa akpa ume (LUAD) na ọrịa kansa squamous cell (LUSC) bụ ụdị ọrịa kansa akpa ume nke na-abụghị obere sel (NSCLC). Ebe ọ bụ na LUSC nwere njikọ siri ike na ojiji ụtaba47 na LUAD bụ ihe a na-ahụkarị na histology nke nodules akpa ume ndị a chọpụtara na nyocha CT48, e wuru ihe nlereanya nkewa anyị kpọmkwem maka ihe atụ nke adenocarcinoma nke ọkwa I. Wang na ndị ọrụ ibe ya lekwasịkwara anya na LUAD ma chọpụta akara lipid itoolu site na iji lipidomics mata ọdịiche dị n'etiti kansa akpa ume na ndị nwere ahụike17. Anyị nwalere ihe nlereanya nkewa ugbu a na ikpe 16 nke LUSC nke ọkwa I na nodules 74 na-adịghị mma ma hụ na amụma LUSC dị ala (AUC 0.776), na-atụ aro na LUAD na LUSC nwere ike inwe akara metabolomic nke ha. N'ezie, egosila na LUAD na LUSC dị iche na etiology, mmalite bayọlọji na mgbanwe mkpụrụ ndụ ihe nketa49. Ya mere, a ga-etinye ụdị histology ndị ọzọ n'ime ụdị ọzụzụ maka nchọpụta nke kansa akpa ume dabere na ọnụọgụgụ mmadụ na mmemme nyocha.
N'ebe a, anyị chọpụtara ụzọ isii kachasị agbanwe agbanwe na adenocarcinoma akpa ume ma e jiri ya tụnyere njikwa ahụike na nodules na-adịghị mma. Xanthine na hypoxanthine bụ ihe ndị a na-ahụkarị na ụzọ metabolism purine. Dịka nsonaazụ anyị si dị, ihe ndị metụtara metabolism purine mụbara nke ukwuu na serum ma ọ bụ anụ ahụ nke ndị ọrịa nwere adenocarcinoma akpa ume ma e jiri ya tụnyere njikwa ahụike ma ọ bụ ndị ọrịa nọ na ọkwa tupu ịwa ahụ15,50. Ọkwa xanthine na hypoxanthine dị elu nke serum nwere ike igosipụta anabolism achọrọ site na ịbawanye mkpụrụ ndụ kansa ngwa ngwa. Nkwụsị nke metabolism glucose bụ ihe ama ama nke metabolism kansa51. N'ebe a, anyị hụrụ mmụba dị ukwuu na pyruvate na lactate na otu LA ma e jiri ya tụnyere otu HC na BN, nke kwekọrọ na akụkọ gara aga nke nsogbu ụzọ glycolytic na profaịlụ metabolome serum nke ndị ọrịa kansa akpa ume na-abụghị obere sel (NSCLC) na njikwa ahụike. nsonaazụ ya na-agbanwe agbanwe52,53.
Ihe dị mkpa bụ na anyị hụrụ njikọ dị n'etiti pyruvate na tryptophan metabolism na serum nke adenocarcinomas akpa ume. E belatara ọkwa tryptophan serum na otu LA ma e jiri ya tụnyere otu HC ma ọ bụ BN. N'ụzọ na-akpali mmasị, otu nnyocha buru ibu gara aga nke ejiri otu ndị nwere ike ịchọta ihe chọpụtara na obere ọkwa nke tryptophan na-agagharị agagharị jikọtara ya na mmụba nke ihe egwu nke kansa akpa ume 54. Tryptophan bụ amino acid dị mkpa nke anyị na-enweta kpamkpam site na nri. Anyị na-ekwubi na mbelata tryptophan serum na adenocarcinoma akpa ume nwere ike igosipụta mbelata ngwa ngwa nke metabolite a. A maara nke ọma na ngwaahịa ikpeazụ nke catabolism tryptophan site na ụzọ kynurenine bụ isi iyi nke de novo NAD+ synthesis. Ebe ọ bụ na a na-emepụta NAD+ karịsịa site na ụzọ mgbapụta, mkpa nke NAD+ na metabolism tryptophan na ahụike na ọrịa ka dị. Nnyocha anyị mere na nchekwa data TCGA gosiri na ngosipụta nke tryptophan transporter solute transporter 7A5 (SLC7A5) mụbara nke ukwuu na adenocarcinoma akpa ume ma e jiri ya tụnyere njikwa nkịtị ma nwee njikọ dị mma na ngosipụta nke glycolytic enzyme GAPDH. Ọmụmụ ihe ndị gara aga lekwasịrị anya karịsịa na ọrụ nke tryptophan catabolism n'igbochi nzaghachi mgbochi ọrịa kansa40,41,42. N'ebe a, anyị na-egosi na igbochi nnabata tryptophan site na ịkụda SLC7A5 na mkpụrụ ndụ kansa akpa ume na-ebute mbelata na ọkwa NAD sel na mbelata nke ọrụ glycolytic n'otu oge. Na nchịkọta, ọmụmụ ihe anyị na-enye ntọala bayọlọji maka mgbanwe na metabolism serum metụtara mgbanwe ọjọọ nke adenocarcinoma akpa ume.
Mgbanwe EGFR bụ mgbanwe kachasị na-akpata mgbanwe ndị ...
Na mmechi, ọmụmụ ihe anyị na-emepụta usoro nhazi metabolic serum maka nchọpụta dị iche iche nke nodules pulmonary ma na-atụ aro usoro ọrụ nke nwere ike ime ka nyocha ihe egwu dịkwuo mma ma mee ka njikwa ahụike dị mfe dabere na nyocha CT scan.
Kọmitii Omume nke Ụlọ Ọgwụ Ọrịa Kansa nke Mahadum Sun Yat-sen, Ụlọ Ọgwụ Mbụ nke Mahadum Sun Yat-sen, na Kọmitii Omume nke Ụlọ Ọgwụ Ọrịa Kansa nke Mahadum Zhengzhou kwadoro ọmụmụ ihe a. N'ime otu nchọpụta na nkwenye dị n'ime, a nakọtara mmadụ 174 sitere na ndị nwere ahụike na mmadụ 244 sitere na nodules benign site n'aka ndị na-eme nyocha ahụike kwa afọ na Ngalaba Nchịkwa na Mgbochi Ọrịa Kansa, Ụlọ Ọrụ Ọrịa Kansa nke Mahadum Sun Yat-sen, na 166 benign nodules. serum. E natara adenocarcinoma akpa ume nke Stage I site na Ụlọ Ọrụ Ọrịa Kansa nke Mahadum Sun Yat-sen. N'ime otu nkwenye mpụga, e nwere ikpe 48 nke nodules benign, ikpe 39 nke adenocarcinoma akpa ume nke stage I site na Ụlọ Ọgwụ Mbụ nke Mahadum Sun Yat-sen, na ikpe 24 nke adenocarcinoma akpa ume nke stage I site na Ụlọ Ọgwụ Kansa nke Zhengzhou. Ụlọ Ọrụ Kansa nke Mahadum Sun Yat-sen chịkọtara ikpe 16 nke kansa akpa ume nke stage I iji nwalee ikike nchọpụta nke nhazi metabolic edobere (egosiri njirimara ndị ọrịa na Tebụl Mgbakwunye 5). A chịkọtara ihe nlele sitere na ndị nchọpụta na ndị nyocha ime n'etiti Jenụwarị 2018 na Mee 2020. A chịkọtara ihe nlele maka ndị nyocha ime n'èzí n'etiti Ọgọst 2021 na Ọktoba 2022. Iji belata ajọ mbunobi nwoke na nwanyị, e kenyere ihe dị ka ọnụọgụgụ ndị ikom na ndị inyom hà nhata na otu ọ bụla. Ndị otu Nchọpụta na Ndị otu Nyocha Ime. E kpebiri okike ndị sonyere dabere na akụkọ onwe onye. E nwetara nkwenye amaara ama site n'aka ndị sonyere niile ma enyeghị ụgwọ ọrụ. Ndị nwere nodules na-adịghị mma bụ ndị nwere akara nyocha CT kwụsiri ike na afọ 2 ruo 5 n'oge nyocha ahụ, ewezuga otu ikpe sitere na ihe nlele nkwenye mpụga, nke a chịkọtara tupu ịwa ahụ ma chọpụta site na histopathology. Ewezuga bronchitis na-adịghị ala ala. A chịkọtara ihe mgbaàmà adenocarcinoma akpa ume tupu a chụpụ akpa ume ma gosi ya site na nchọpụta ọrịa. A chịkọtara ihe nlele ọbara ibu ọnụ na tubes nkewa serum na-enweghị ihe mgbochi ọbara ọ bụla. A kpụkọrọ ihe nlele ọbara maka otu awa na okpomọkụ ụlọ wee kpụọ ya na 2851 × g maka nkeji 10 na 4°C iji chịkọta ihe mkpuchi serum. E tinyere mkpụrụ ọgwụ ndị dị n'ime serum n'ime -80°C ruo mgbe ewepụtara ha metabolites. Ngalaba Mgbochi Ọrịa Kansa na Nnyocha Ahụike nke Ụlọ Ọrụ Ọrịa Kansa nke Mahadum Sun Yat-sen chịkọtara otu mkpụrụ ọgwụ serum n'aka mmadụ 100 nwere ahụike, gụnyere ụmụ nwoke na ụmụ nwanyị dị afọ 40 ruo 55 hà nhata. E gwakọtara nha nhata nke ihe nlele ọ bụla nke onye nyere onyinye, e kewara ya ma chekwaa ya na -80°C. E jiri ngwakọta serum ahụ mee ihe dị ka ihe ntụaka maka njikwa mma na nhazi data.
E gbazee ihe nlele serum na ule ahụ ma wepụta metabolites site na iji usoro mwepụta jikọtara ọnụ (MTBE/methanol/mmiri) 56. N'ụzọ dị mkpirikpi, e tinyere 50 μl nke serum na 225 μl nke methanol oyi-ọcha na 750 μl nke methyl tert-butyl ether (MTBE). Gwakọta ngwakọta ahụ ma tinye ya na ice ruo otu awa. E wee gwakọta ihe nlele ndị ahụ ma gwakọta ha na 188 μl nke mmiri MS-grade nwere ụkpụrụ dị n'ime (13C-lactate, 13C3-pyruvate, 13C-methionine, na 13C6-isoleucine, nke e zụtara na Cambridge Isotope Laboratories). E wee tinye ngwakọta ahụ na centrifuge na 15,000 × g maka nkeji iri na 4 °C, e wee bufee usoro dị ala n'ime tube abụọ (125 μL nke ọ bụla) maka nyocha LC-MS na ụdị dị mma na nke na-adịghị mma. N'ikpeazụ, a kpọnwụrụ ihe nlele ahụ ruo mgbe ọ kpọrọ nkụ n'ime ihe na-eme ka ikuku dị elu.
E tinyere metabolites ndị a kpọrọ nkụ n'ime 120 μl nke 80% acetonitrile, gbanye ha ọkụ ruo nkeji ise, ma tinye ha na centrifuge na 15,000 × g maka nkeji iri na 4°C. E tinyere supernatants n'ime vials amber glass vials na microinserts maka ọmụmụ ihe gbasara metabolomics. Nyocha metabolomics na-enweghị ebumnuche na ikpo okwu ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). E kewapụrụ Metabolites site na iji sistemụ Dionex Ultimate 3000 UPLC na kọlụm ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Waters). Na ọnọdụ ion dị mma, usoro mkpanaka bụ 95% (A) na 50% acetonitrile (B), nke ọ bụla nwere 10 mmol/L ammonium acetate na 0.1% formic acid. N'ọnọdụ adịghị mma, usoro mkpanaka A na B nwere 95% na 50% acetonitrile, n'otu n'otu, usoro abụọ ahụ nwere 10 mmol/L ammonium acetate, pH = 9. Mmemme gradient bụ dịka 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. E jigide kọlụm ahụ na 40°C na ihe nlele ahụ na 10°C na ihe nlele akpaaka. Ọnụego mmiri ahụ bụ 0.3 ml/min, olu ntụtụ bụ 3 μl. E jiri Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) nke nwere isi iyi electrospray ionization (ESI) rụọ ọrụ na ụdị nyocha zuru oke ma jikọta ya na ụdị nlekota ddMS2 iji chịkọta nnukwu data. E debere paramita MS dịka ndị a: voltaji ịgba +3.8 kV/- 3.2 kV, okpomọkụ capillary 320°C, gas nchekwa 40 arb, gas enyemaka 10 arb, okpomọkụ ihe na-ekpo ọkụ probe 350°C, oke nyocha 70–1050 m/h, mkpebi. 70 000. E ji Xcalibur 4.1 (Thermo Fisher Scientific) nweta data.
Iji chọpụta ogo data, e mepụtara ihe nlele njikwa mma (QC) achịkọtara site na iwepụ ihe dị ka 10 μL nke ihe na-asọpụ n'ime ihe nlele ọ bụla. E nyochachara ihe nlele njikwa mma isii na mmalite usoro nyocha iji chọpụta nkwụsi ike nke sistemụ UPLC-MS. A na-ewebata ihe nlele njikwa mma n'ime otu ahụ mgbe niile. LC-MS nyochachara otu 11 nke ihe nlele serum dị na ọmụmụ a. Ejiri ihe nchịkọta nke ngwakọta ọdọ mmiri serum sitere na ndị na-enye onyinye 100 dị mma mee ihe dị ka ihe ntụaka n'otu n'otu iji nyochaa usoro mwepụta ma gbanwee maka mmetụta ogbe-site-otu. E mere nyocha metabolomics na-enweghị ebumnuche nke otu nchọpụta, otu nkwenye dị n'ime, na otu nkwenye mpụga na Metabolomics Center nke Mahadum Sun Yat-sen. Ụlọ nyocha mpụga nke Guangdong University of Technology Analysis and Testing Center nyochakwara ihe nlele 40 sitere na otu mpụga iji nwalee arụmọrụ nke ihe nlereanya nkewa.
Mgbe ewepụtara ma gbanwee ya, a tụrụ ọnụọgụ zuru oke nke metabolites serum site na iji ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) yana isi iyi electrospray ionization (ESI) na ọnọdụ nlekota mmeghachi omume dị iche iche (MRM). Ejiri kọlụm ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Mmiri) kewaa metabolites. Usoro mkpanaka nwere 90% (A) na 5% acetonitrile (B) yana 10 mmol/L ammonium acetate na 0.1% ngwọta ammonia. Mmemme gradient bụ dịka ndị a: 0–1.5 nkeji, 0% B; 1.5–6.5 nkeji, 0–15% B; 6.5–8 nkeji, 15% B; 8–8.5 nkeji, 15%–0% B; 8.5–11.5 nkeji, 0% B. A nọgidere na-ejigide kọlụm ahụ na 40 °C na ihe nlele ahụ na 10 °C na ihe nlele ahụ. Ọkwa mmiri ahụ bụ 0.3 mL/min, olu nwụnye ahụ bụ 1 μL. E debere paramita MS dịka ndị a: voltaji capillary ±3.5 kV, nrụgide nebulizer 35 psi, mmiri gas sheath 12 L/min, okpomọkụ gas sheath 350°C, okpomọkụ gas kpọrọ nkụ 250°C, na mmiri gas akpọ nkụ 14 l/min. Mgbanwe 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. E ji Mass Hunter B.07.00 (Agilent Technologies) chịkọta data. Maka ihe nlele serum, e jiri usoro nhazi nke ngwọta ngwakọta ọkọlọtọ tụọ tryptophan, pyruvate, lactate, hypoxanthine, na xanthine. Maka ihe nlele sel, a na-eme ka ọdịnaya tryptophan dị ka ọkọlọtọ dị n'ime na oke protein sel si dị.
E jiri Compound Discovery 3.1 na TraceFinder 4.0 (Thermo Fisher Scientific) mee mwepụta kacha elu (m/z na oge nchekwa (RT)). Iji wepụ ọdịiche dị n'etiti ogbe, e kewara elu njirimara ọ bụla nke ihe nlele ule site na elu njirimara nke ihe ntụaka site na otu ogbe ahụ iji nweta oke nha. Egosiputara ọdịiche ọkọlọtọ nke ọkọlọtọ dị n'ime tupu na mgbe nhazi ahụ gasịrị na Tebụl Mgbakwunye 6. E ji ọnụego nchọpụta ụgha mara ọdịiche dị n'etiti otu abụọ ahụ (FDR <0.05, ule ọkwa Wilcoxon bịanyere aka na ya) na mgbanwe mpịachi (>1.2 ma ọ bụ <0.83). E gosipụtara data MS dị ụkọ nke atụmatụ ewepụtara na data MS edoziri serum na Data Mgbakwunye 1 na Data Mgbakwunye 2, n'otu n'otu. Emere nkọwa kacha elu dabere na ọkwa njirimara anọ akọwapụtara, gụnyere metabolites achọpụtara, ogige ndị a kọwara nke ọma, klas ndị mejupụtara, na ogige ndị a na-amaghị ama 22. Dabere na nchọta nchekwa data na Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), e mechara họrọ ngwakọta ndị dị ndụ nwere ọkọlọtọ MS/MS ma ọ bụ nkọwa kpọmkwem na mzCloud (akara > 85) ma ọ bụ Chemspider dị ka ndị na-eme n'etiti metabolome dị iche. Agụnyere nkọwa kacha elu maka atụmatụ ọ bụla na Data Mgbakwunye 3. Ejiri MetaboAnalyst 5.0 mee nyocha univariate nke ọnụọgụ metabolite zuru oke. MetaboAnalyst 5.0 nyochakwara nyocha mmụba ụzọ KEGG dabere na metabolites dị iche iche. Ejiri ngwugwu ngwanrọ ropls (v.1.26.4) nyochaa nyocha akụkụ (PCA) na nyocha discriminant nke akụkụ pere mpe (PLS-DA). Ejiri ngwugwu ngwanrọ ropls (v.1.26.4) nyochaa nhazi stack na autoscaling. Ejiri usoro biomarker metabolite kacha mma mepụta ihe nlereanya biomarker metabolite kacha mma maka ịkọ amụma ọrịa nodule site na iji regression binary logistic na onye ọrụ nhọrọ kacha nta (LASSO, ngwugwu R v.4.1-3). A kọwara arụmọrụ nke ụdị dị iche iche na nhazi nchọpụta na nkwenye site na ịtụ AUC dabere na nyocha ROC dịka ngwugwu pROC si dị (v.1.18.0.). E nwetara njedebe kacha mma nke ohere dabere na ntinye Youden kachasị elu nke ụdị ahụ (nsogbu + nkọwapụta - 1). A ga-ebu amụma ihe nlele nwere ụkpụrụ dị ala ma ọ bụ karịa karịa oke dị ka nodules na-adịghị mma na adenocarcinoma akpa ume, n'otu n'otu.
A kụrụ mkpụrụ ndụ A549 (#CCL-185, Nchịkọta Omenala nke Ndị America) na F-12K nke nwere 10% FBS. E tinyere usoro RNA dị mkpụmkpụ (shRNA) nke na-elekwasị anya na SLC7A5 na njikwa na-abụghị ihe mgbaru ọsọ (NC) n'ime vector lentiviral pLKO.1-puro. Usoro antisense nke shSLC7A5 bụ ndị a: Sh1 (5′-GGAGAAACCTGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGAACTTAT-3′). E zụtara ọgwụ mgbochi SLC7A5 (#5347) na tubulin (#2148) site na Teknụzụ Cell Signaling. E jiri ọgwụ mgbochi SLC7A5 na tubulin mee ihe na ngwakọta nke 1:1000 maka nyocha Western blot.
Nnwale Nrụgide Seahorse XF Glycolytic na-atụle ọkwa acidification extracellular (ECAR). N'ime nnwale ahụ, e nyere glucose, oligomycin A, na 2-DG n'usoro iji nwalee ikike glycolytic nke sel dịka ECAR si tụọ.
E tinyere mkpụrụ ndụ A549 e tinyere ihe na-anaghị elekwasị anya (NC) na shSLC7A5 (Sh1, Sh2) n'ime efere dị sentimita iri n'obosara. E jiri 1 ml nke methanol mmiri oyi 80% wepụta mkpụrụ ndụ ndị dị na mmiri methanol, chịkọta ha n'ime tube ọhụrụ, wee kpụọ ha na centrifuge na 15,000 × g maka nkeji iri na ise na 4°C. Chịkọta 800 µl nke ihe na-asọ asọ ma kpọọ nkụ site na iji ihe na-eme ka ikuku dị elu kpoo ha. E jiri LC-MS/MS dịka akọwara n'elu tụọ ọkwa NAD(H) nke mkpụrụ ndụ na mkpụrụ ndụ A549 (NC na shSLC7A5) dịka ntuziaka onye nrụpụta si dị. A tụrụ ọkwa protein maka ihe nlele ọ bụla iji mee ka ọnụọgụ metabolites dị mma.
E jirighị usoro ọnụọgụgụ mee ihe iji chọpụta nha ihe nlele ahụ tupu oge eruo. A tụlere ọmụmụ ihe metabolomics gara aga nke lekwasịrị anya na nchọpụta biomarker15,18 dị ka ihe nrịbama maka mkpebi nha, ma e jiri ya tụnyere akụkọ ndị a, ihe nlele anyị zuru oke. E wepụghị ihe nlele ọ bụla na otu ọmụmụ ihe ahụ. E kenyere ihe nlele serum na otu nchọpụta (ikpe 306, 74.6%) na otu nkwenye dị n'ime (ikpe 104, 25.4%) maka ọmụmụ ihe metabolomics na-enweghị ebumnuche. Anyị họrọkwara ikpe 70 site na otu ọ bụla site na ntọala nchọpụta maka ọmụmụ ihe metabolomics ezubere iche. Ndị nyocha ahụ kpuru ìsì ịhụ ọrụ otu n'oge nchịkọta na nyocha data LC-MS. A kọwara nyocha ọnụọgụgụ nke data metabolomics na nnwale sel na ngalaba Nsonaazụ, Akụkọ Ihe Osise, na Usoro dị iche iche. Emere ọnụọgụgụ nke ọrụ tryptophan cell, NADT, na glycolytic ugboro atọ n'adabereghị onwe ya yana nsonaazụ yiri ya.
Maka ozi ndị ọzọ gbasara nhazi ọmụmụ ihe a, lee Nchịkọta Akụkọ Nchịkọta Ihe Okike nke metụtara akụkọ a.
E gosiri data MS raw nke atụmatụ ewepụtara na data MS a haziri nke ọma nke serum ntụaka na Data Mgbakwunye 1 na Data Mgbakwunye 2, n'otu n'otu. E gosipụtara nkọwa kachasị elu maka atụmatụ dị iche iche na Data Mgbakwunye 3. Enwere ike ibudata data LUAD TCGA na https://portal.gdc.cancer.gov/. E nyere data ntinye maka ịkọ eserese ahụ na data isi mmalite. E nyere data isi mmalite maka akụkọ a.
Òtù Ọmụmụ Ihe Nnyocha Agụụ nke Mba, wdg. Ibelata ọnwụ nke kansa akpa ume site na iji obere dose nke kọmputa na-eme ihe. Northern England. J. Med. 365, 395–409 (2011).
Kramer, BS, Berg, KD, Aberle, DR na Prophet, PC Nnyocha kansa akpa ume site na iji obere dose nke helical CT: nsonaazụ sitere na Ọmụmụ Nyocha Akpa Mba (NLST). J. Med. Ihuenyo 18, 109–111 (2011).
De Koning, HJ, na ndị ọzọ. Ibelata ọnwụ nke kansa akpa ume site na nyocha CT volumetric na nnwale enweghị usoro. Northern England. J. Med. 382, 503–513 (2020).
Oge ozi: Sep-18-2023