Immunomodulatory metabolites yɛ ade titiriw a ɛwɔ tumor microenvironment (TME) no mu, nanso sɛ kakraa bi pɛ a, wonnim wɔn dodow no ara. Ɛha yi, yɛhwehwɛɛ akisikuru ne T nkwammoaa a efi ayarefo a wɔwɔ high-grade serous carcinoma (HGSC) akisikuru ne ascites mu mu de daa metabolome a ɛwɔ TME adan ahorow yi mu adi. Ascites ne tumor nkwammoaa wɔ nsonsonoe kɛse wɔ metabolite mu. Sɛ wɔde toto ascites ho a, 1-methylnicotinamide (MNA) dɔɔso kɛse wɔ T nkwammoaa a ɛhyɛn akisikuru mu no mu. Ɛwom sɛ MNA dodow a ɛwɔ T nkwammoaa mu no kɔ soro de, nanso nicotinamide N-methyltransferase (enzyme a ɛma methyl akuw ahorow fi S-adenosylmethionine kɔ nicotinamide mu) a wɔda no adi no yɛ fibroblasts ne tumor nkwammoaa nkutoo. Wɔ dwumadi mu no, MNA ma T nkwammoaa no ma cytokine tumor necrosis factor alpha a ɛma akisikuru no yɛ kɛse no fi mu. Enti, MNA a wonya fi TME mu no boa ma T nkwammoaa no tumi a ɛko tia nyarewa no yɛ adwuma na egyina hɔ ma nipadua no mu tumi a ɛko tia nyarewa no botae a ebetumi ayɛ de asa nnipa kokoram.
Nneɛma a ɛma nipadua no yɛ adwuma a efi akisikuru mu no betumi anya tumi a ɛko tia akisikuru no so nkɛntɛnso kɛse, na adanse pii kyerɛ sɛ ebetumi nso ayɛ tumi titiriw a ɛkanyan yare no nkɔso (1). Wɔ Warburg nkɛntɛnso no akyi no, wɔafi ase ayɛ adwuma nnansa yi de akyerɛ sɛnea akisikuru nkwammoaa no yɛ adwuma wɔ nipadua mu ne abusuabɔ a ɛda akisikuru no mu nneɛma a ɛko tia nyarewa tebea (TME) no ntam. Nhwehwɛmu ahorow a wɔayɛ wɔ mmoadabi nhwɛso ne nnipa T nkwammoaa ho no ada no adi sɛ glutamine a ɛsakra (2), oxidative metabolism (3) ne glucose metabolism (4) betumi ayɛ adwuma wɔ ahofadi mu wɔ nipadua no mu nkwammoaa a ɛko tia nyarewa akuw nketewa ahorow so. Nneɛma pii a ɛma nipadua no yɛ adwuma wɔ saa akwan yi mu no siw T nkwammoaa no dwumadi a ɛko tia akisikuru no ano. Wɔada no adi sɛ coenzyme tetrahydrobiopterin (BH4) a wosiw ano no betumi asɛe T nkwammoaa a ɛdɔɔso, na BH4 a ɛkɔ soro wɔ nipadua no mu no betumi ama nipadua no mu tumi a ɛko tia akisikuru a CD4 ne CD8 yɛ ntamgyinafo no ayɛ kɛse. Afei nso, wobetumi agye kynurenine tumi a ɛma nipadua no tumi ko tia nyarewa no denam BH4 a wɔde bɛma so (5). Wɔ isocitrate dehydrogenase (IDH) mutant glioblastoma mu no, enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) a wɔde sie no siw T nkwammoaa dwumadi, dodow ne cytolysis Dwumadi (6) ano. Nnansa yi, wɔada no adi sɛ methylglyoxal, ade a efi glycolysis mu ba no, suppressor cells a efi myeloid mu na ɛyɛ, na T cell transfer of methylglyoxal betumi asiw effector T cell dwumadi ano. Wɔ ayaresa mu no, methylglyoxal a wɔma ɛnyɛ hwee no betumi adi myeloid-derived suppressor cells (MDSC) dwumadi so na ama checkpoint blockade ayaresa no ayɛ kɛse wɔ mmoadabi nhwɛso ahorow mu ( 7 ). Saa nhwehwɛmu ahorow yi nyinaa si dwuma titiriw a nneɛma a ɛma nipadua no yɛ adwuma a wonya fi TME mu di wɔ T nkwammoaa dwumadi ne dwumadi a wɔhwɛ so no so dua.
Wɔabɔ amanneɛ kɛse sɛ T nkwammoaa ntumi nyɛ adwuma yiye wɔ awotwaa mu kokoram mu (8). Eyi fã bi fi nipadua mu nneɛma a ɛsakra a ɛwɔ hypoxia ne akisikuru ntini a ɛnteɛ mu (9), a ɛma glucose ne tryptophan dan nneɛma a ɛka ho te sɛ lactic acid ne kynurenine. Lactate a ɛboro so a ɛwɔ nkwammoaa no akyi no brɛ interferon-γ (IFN-γ) a wɔyɛ no ase na ɛma nsonsonoe ba myelosuppressive akuw nketewa mu ( 10 , 11 ). Tryptophan a wɔnom no siw T nkwammoaa a ɛdɔɔso no ano tẽẽ na esiw T nkwammoaa agyefo sɛnkyerɛnne kwan so (12-14). Ɛmfa ho saa nsɛm yi a wɔahu no, wɔyɛɛ adwuma pii a ɛfa nipadua no mu tumi a ɛko tia nyarewa ho wɔ in vitro T cell culture mu denam media a wɔayɛ no yiye so, anaasɛ wɔde too hɔ wɔ homologous mouse models in vivo mu, a emu biara nkyerɛ sɛnea nnipa kokoram ne Physiological macro ne micro environment no yɛ soronko koraa.
Ade a ɛtaa ba wɔ awotwaa mu kokoram ho ne sɛnea ɛtrɛw wɔ awotwaa mu na ɛma ascites ba. Nkwammoaa mu nsu a ɛboaboa ano wɔ ascites mu no ne yare a ɛkɔ anim ne nkɔmhyɛ a enye wɔ abusuabɔ ( 15 ). Sɛnea amanneɛbɔ kyerɛ no, saa dan soronko yi yɛ hypoxic, ɛwɔ vascular endothelial growth factor (VEGF) ne indoleamine 2,3-dioxygenase (IDO) pii, na T regulatory cells ne myeloid inhibitory cells na ɛhyɛn mu (15-18). Ebia ɛsono sɛnea ascites nipadua no yɛ adwuma no yɛ soronko wɔ akisikuru no ankasa de ho, enti T nkwammoaa a wɔsan yɛ nhyehyɛe wɔ peritoneal space no mu no mu nna hɔ. Afei nso, nsonsonoe titiriw ne nsonsonoe a ɛda ascites ne metabolites a ɛwɔ akisikuru no mu ntam no betumi asiw nkwammoaa a ɛko tia nyarewa no a ɛbɛkɔ mu ne sɛnea ɛyɛ adwuma wɔ akisikuru so no kwan, na nhwehwɛmu foforo ho hia.
Sɛnea ɛbɛyɛ a yebedi ɔhaw ahorow yi ho dwuma no, yɛyɛɛ sensitive cell separation and liquid chromatography tandem mass spectrometry (LC-MS/MS) kwan a yɛde besua nkwammoaa ahorow ahorow (a CD4 + ne CD8 + T nkwammoaa ka ho) ne afei nso wɔ akisikuru mu ne ntam Ne metabolites span nkwammoaa a ɛwɔ ascites ne tumor tebea koro no ara mu wɔ ɔyarefo no mu. Yɛde saa kwan yi di dwuma de ka high-dimensional flow cytometry ne single-cell RNA sequencing (scRNA-seq) ho de ma mfonini a wɔasiesie no yiye a ɛkyerɛ sɛnea saa nnipa atitiriw yi metabolic tebea. Saa kwan yi daa nkɔanim kɛse adi wɔ 1-methylnicotinamide (MNA) dodow a ɛwɔ tumor T nkwammoaa mu, na nhwehwɛmu ahorow a wɔyɛe wɔ vitro kyerɛe sɛ kan no na wonnim tumi a MNA nya wɔ T nkwammoaa dwumadi so wɔ nipadua no mu tumi a ɛko tia nyarewa no. Mpɛn pii no, saa kwan yi da nkitahodi a ɛda akisikuru ne nipadua mu nkwammoaa a ɛko tia nyarewa ntam adi, na ɛma yenya nhumu soronko wɔ nipadua no mu ahoɔden a ɛko tia nyarewa no mu nneɛma a ɛko tia nyarewa ho, a ebia mfaso wɔ so ma T nkwammoaa a egyina awotwaa mu kokoram so ayaresa a ɛko tia nyarewa no ayaresa Ayaresa hokwan ahorow.
Yɛde high-dimensional flow cytometry dii dwuma de kyerɛɛ glucose a wɔfa bere koro mu dodow [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) ne mitochondrial dwumadi [MitoTracker Deep Red (MT DR)] (7, 19, 20) wɔ nkyɛnkyɛn typical agyiraehyɛde ahorow a ɛma nsonsonoe da nkwammoaa a ɛko tia nyarewa ne akisikuru nkwammoaa dodow mu (Table S2 ne Mfonini S1A). Saa nhwehwɛmu yi kyerɛe sɛ sɛ wɔde toto T nkwammoaa ho a, ascites ne tumor nkwammoaa wɔ glucose a wɔfa no kɛse, nanso nsonsonoe nketenkete wɔ mitochondrial dwumadi mu. Sɛ wɔkyekyem pɛpɛɛpɛ a, glucose a tumor nkwammoaa [CD45-EpCAM (EpCAM)+] gye no yɛ T nkwammoaa mmɔho abiɛsa kosi anan, na sɛ wɔkyekyem pɛpɛɛpɛ a, glucose a CD4 + T nkwammoaa gye no yɛ CD8 + T nkwammoaa mmɔho 1.2, a ɛkyerɛ sɛ Tumor infiltrating lymphocytes (TIL) wɔ nipadua mu ahwehwɛde ahorow wɔ TME koro no ara mu mpo (Mfonini 1A). Nea ɛne eyi bɔ abira no, mitochondrial dwumadi a ɛwɔ akisikuru nkwammoaa mu no te sɛ CD4 + T nkwammoaa no, na mitochondrial dwumadi a ɛwɔ nkwammoaa ahorow abien no nyinaa mu no korɔn sen CD8 + T nkwammoaa no de (Mfonini 1B). Mpɛn pii no, nea efi mu ba yi da nipadua mu nneɛma a ɛsakra no adi. Akisikuru nkwammoaa no nipadua mu nneɛma a ɛsakra no korɔn sen CD4 + T nkwammoaa no, na CD4 + T nkwammoaa no nipadua mu nneɛma a ɛsakra no korɔn sen CD8 + T nkwammoaa no de. Ɛmfa ho sɛ saa nsunsuanso yi wɔ nkwammoaa ahorow mu no, nsonsonoe biara nni CD4 + ne CD8 + T nkwammoaa no nipadua mu nneɛma a ɛsakra tebea mu anaa wɔn dodow a ɛfa ho wɔ ascites mu bere a wɔde toto akisikuru ho no mu (Mfonini 1C). Nea ɛne eyi bɔ abira no, wɔ CD45-nkwaboaa fã no mu no, EpCAM + nkwammoaa dodow a ɛwɔ akisikuru no mu no kɔɔ soro bere a wɔde totoo ascites ho no (Mfonini 1D). Yɛsan nso huu nsonsonoe a ɛda adi pefee wɔ nipadua mu nneɛma mu wɔ EpCAM + ne EpCAM- nkwammoaa mu nneɛma ntam. EpCAM + (tumor) nkwammoaa wɔ glucose a wɔfa ne mitochondrial dwumadi kɛse sen EpCAM- nkwammoaa, a ɛkorɔn sen fibroblasts a ɛwɔ akisikuru nkwammoaa mu wɔ TME mu no metabolic dwumadi (Mfonini 1, E ne F).
(A ne B) Mfinimfini fluorescence ahoɔden (MFI) a glucose a wɔfa (2-NBDG) (A) ne mitochondrial dwumadi a CD4 + T nkwammoaa (MitoTracker kɔkɔɔ tuntum) (B) Ananmusifo graphs (benkum) ne tabulated data (Nifa), CD8 + T nkwammoaa ne EpCAM + CD45-akisikuru nkwammoaa a efi ascites ne tumor. (C) CD4 + ne CD8 + nkwammoaa (a ɛwɔ CD3 + T nkwammoaa mu) a ɛwɔ ascites ne tumor mu no nsusuwii. (D) EpCAM + akisikuru nkwammoaa no fã bi wɔ ascites ne akisikuru mu (CD45−). (E ne F) EpCAM + CD45-akisikuru ne EpCAM-CD45-matrix glucose a wɔfa (2-NBDG) (E) ne mitochondrial dwumadi (MitoTracker kɔkɔɔ tuntum) (F) ananmusifo graphs (benkum) ne tabulated data ( Nifa) Ascites ne tumor nkwammoaa. (G) CD25, CD137 ne PD1 a wɔda no adi denam flow cytometry so ho mfonini ahorow a ɛyɛ ananmusifo. (H ne I) CD25, CD137 ne PD1 a wɔda no adi wɔ CD4 + T nkwammoaa (H) ne CD8 + T nkwammoaa (I) so. (J ne K) Naive, central memory (Tcm), effector (Teff) ne effector memory (Tem) phenotypes a egyina CCR7 ne CD45RO a wɔda no adi so. Mfonini ahorow a egyina hɔ ma (benkum so) ne tabular data (nifa so) a ɛkyerɛ CD4 + T nkwammoaa (J) ne CD8 + T nkwammoaa (K) a ɛwɔ ascites ne tumors mu. P values ​​a wɔde paired t-test (* P <0.05, **P <0.01 ne ***P <0.001) na ɛkyerɛ. Ntrɛwmu no gyina hɔ ma ayarefo a wɔayɛ pɛ (n = 6). FMO, fluorescence a wɔayi biako afi mu; MFI, mfinimfini fluorescence ahoɔden.
Nhwehwɛmu foforo daa nsonsonoe titiriw afoforo adi wɔ T nkwammoaa phenotypic tebea a wɔasiesie no kɛse no ntam. Activated (Mfonini 1, G kosi I) ne effector memory (Mfonini 1, J ne K) a ɛwɔ akisikuru mu no taa ba kɛse sen ascites (CD3 + T nkwammoaa fã). Saa ara nso na nhwehwɛmu a wɔyɛe wɔ phenotype no mu denam activation markers (CD25 ne CD137) ne depletion markers [programmed cell death protein 1 (PD1)] a wɔda no adi so no kyerɛe sɛ ɛwom sɛ ɛsono saa nnipa dodow yi nipadua mu nneɛma a ɛsakra no su (Mfonini S1, B kosi E) , Nanso wɔanhu nsonsonoe titiriw biara wɔ nipadua mu nneɛma mu bere nyinaa wɔ naive, effector anaa memory subsets ntam (Mfonini S1, F kosi I). Wɔnam mfiri a wɔde sua ade akwan a wɔfaa so de nkwammoaa mu phenotypes (21) maa wɔn ankasa so sii nea efii mu bae yi so dua, na ɛno san daa no adi sɛ nnompe mu nkwammoaa dodow bi (CD45 + / CD3- / CD4 + / CD45RO +) wɔ ɔyarefo no ascites mu (Mfonini S2A ). Wɔ nkwammoaa ahorow a wɔakyerɛ no nyinaa mu no, saa myeloid nkwammoaa dodow yi daa glucose a wɔfa ne mitochondrial dwumadi a ɛsen biara adi (Mfonini S2, B kosi G). Nea afi mu aba yi si nsonsonoe a emu yɛ den a ɛwɔ nipadua mu nneɛma a ɛsakra mu wɔ nkwammoaa ahorow pii a wohu wɔ ascites ne tumors mu wɔ HGSC ayarefo mu no so dua.
Asɛnnennen titiriw a ɛwɔ TIL metabolic su ahorow ntease mu ne hia a ehia sɛ wɔyi T nkwammoaa nhwɛsode a ɛho tew, ne su ne dodow a ɛfata fi akisikuru mu. Nnansa yi nhwehwɛmu ada no adi sɛ akwan a wɔfa so yiyi nneɛma mu na ɛma nhwiren yɛ kɛse a egyina flow cytometry so no betumi ama nsakrae aba nkwammoaa mu metabolite profiles mu ( 22-24 ). Sɛnea ɛbɛyɛ na yɛadi ɔhaw yi so no, yɛyɛɛ bead enrichment kwan no yiye de yii TIL fii nnipa awotwaa mu kokoram a wɔatwa no oprehyɛn mu na yɛayi afi mu ansa na wɔde LC-MS/MS ayɛ nhwehwɛmu (hwɛ Nneɛma ne Akwan; Mfonini 2A). Sɛnea ɛbɛyɛ a yɛbɛhwɛ nkɛntɛnso a saa nhyehyɛe yi nya wɔ metabolite nsakrae so nyinaa no, yɛde metabolite profiles a ɛwɔ T nkwammoaa a wɔde mae a wɔwɔ apɔwmuden de mae wɔ atifi hɔ bead mpaapaemu anammɔn no akyi no totoo nkwammoaa a wɔanpaapae bead mu nanso wɔtraa nsukyenee so no ho. Saa nhwehwɛmu a ɛfa su pa ho yi hunuu sɛ abusuabɔ kɛseɛ wɔ saa tebea mmienu yi ntam (r = 0.77), na mfiridwuma mu repeatability a ɛwɔ metabolites 86 kuw no mu no wɔ repeatability kɛseɛ (Mfonini 2B). Enti, saa akwan yi betumi ayɛ metabolite nhwehwɛmu a edi mu wɔ nkwammoaa a wɔreyɛ cell type enrichment mu, na ɛnam so ama wɔanya platform a edi kan a ɛwɔ nsusuwii a ɛkorɔn a wɔde hu metabolites pɔtee bi wɔ HGSC mu, na ɛnam so ama nkurɔfo anya ntease a emu dɔ wɔ nkwammoaa pɔtee bi ho Sexual metabolism program.
(A) Schematic mfonini a ɛkyerɛ magnetic bead enrichment. Ansa na wɔde LC-MS/MS bɛyɛ nhwehwɛmu no, wɔbɛfa magnetic bead enrichment mprɛnsa a ɛtoatoa so anaasɛ wɔbɛkɔ so atra nsukyenee so. (B) Nkɛntɛnso a enrichment type nya wɔ metabolites dodow so. Sɛ wɔkyekyɛ mu a, susudua abiɛsa ma enrichment type biara ± SE. Ntrɛwmu a ɛyɛ fitaa no gyina hɔ ma abusuabɔ a ɛyɛ 1:1. Intra-class correlation (ICC) a ɛfa susudua a wɔsan yɛ no mpɛn pii a wɔakyerɛ wɔ axis label no mu. NAD, nicotinamide a wɔde yɛ aduru a wɔfrɛ no adenine dinucleotide. (C) Schematic diagram a ɛkyerɛ adwumayɛ kwan a wɔfa so yɛ ayarefo metabolite nhwehwɛmu. Wɔboaboa ascites anaa akisikuru ano fi ayarefo hɔ na wɔkora so wɔ cryopreserved mu. Wɔde flow cytometry yɛɛ nhwɛsode biara fã ketewaa bi mu nhwehwɛmu, bere a wɔde nhwɛsode a aka no yɛɛ enrichment mprɛnsa maa CD4+, CD8+ ne CD45- nkwammoaa. Wɔde LC-MS/MS yɛɛ nkwammoaa nketenkete yi mu nhwehwɛmu. (D) Ɔhyew ho mfonini a ɛkyerɛ sɛnea metabolite dodow a wɔahyɛ da ayɛ. Dendrogram no gyina hɔ ma Ward a ɔboaboaa Euclidean akwansin a ɛda nhwɛsode ahorow ntam no ano. (E) Principal component analysis (PCA) of sample metabolite map, a ɛkyerɛ nhwɛsode biara a wɔasan ayɛ abiɛsa, nhwɛsode a efi ɔyarefo koro no ara mu no wɔde nhama bi abɔ mu. (F) PCA a ɛfa metabolite profile a ɛwɔ nhwɛsode no tebea mu wɔ ɔyarefo no so (kyerɛ sɛ, wɔde partial redundancy di dwuma); nhwɛsode no su no anohyeto wɔ convex hull no mu. PC1, ade titiriw 1; PC2, ade titiriw 2.
Afei, yɛde saa enrichment kwan yi dii dwuma de hwehwɛɛ metabolites 99 a ɛwɔ CD4 +, CD8 + ne CD45-cell fractions mu wɔ ascites ne tumors titiriw a ɛwɔ HGSC ayarefo baanum mu (Mfonini 2C, Mfonini S3A ne Table S3 ne S4). Nnipa dodow a wɔn ani gye ho no yɛ 2% kosi 70% wɔ mfitiase nkwammoaa a nkwa wom nhwɛsode kɛse no mu, na nkwammoaa no fã gu ahorow kɛse wɔ ayarefo ntam. Sɛ wɔtetew nhwiren no mu wie a, sɛ wɔkyekyem pɛpɛɛpɛ a, anigye fã a wɔahyɛ no den (CD4+, CD8+ anaa CD45-) no yɛ bɛboro 85% wɔ nkwammoaa a nkwa wom a ɛwɔ nhwɛsode no mu nyinaa mu. Saa kwan a wɔfa so ma nneɛma yɛ kɛse yi ma yetumi hwehwɛ nkwammoaa dodow mu fi nnipa akisikuru ntini mu nneɛma a ɛsakra mu, a ɛrentumi nyɛ yiye mfi nhwɛsode akɛse mu. Yɛde saa nhyehyɛe yi dii dwuma no, yehui sɛ l-kynurenine ne adenosine, saa immunosuppressive metabolites abien a wɔakyerɛkyerɛ mu yiye yi kɔɔ soro wɔ tumor T nkwammoaa anaa tumor nkwammoaa mu (Mfonini S3, B ne C). Enti, nea afi mu aba yi kyerɛ nokwaredi ne tumi a yɛn nkwammoaa mu mpaapaemu ne mass spectrometry mfiridwuma no tumi hu nneɛma a ɛho hia wɔ abɔde mu wɔ ayarefo ntini mu.
Yɛn nhwehwɛmu no nso daa metabolic mpaapaemu a emu yɛ den adi wɔ nkwammoaa ahorow a ɛwɔ ayarefo mu ne wɔn ntam (Mfonini 2D ne Mfonini S4A). Titiriw no, sɛ wɔde toto ayarefo afoforo ho a, ɔyarefo 70 daa nipadua mu nneɛma a ɛsakra no su ahorow adi (Mfonini 2E ne Mfonini S4B), a ɛkyerɛ sɛ ebia nipadua mu nneɛma a ɛsakra no mu nsonsonoe kɛse wɔ ayarefo no ntam. Ɛfata sɛ yɛhyɛ no nsow sɛ sɛ wɔde toto ayarefo afoforo ho (lita 1.2 kosi 2; Table S1) a, na ascites dodow a wɔboaboaa ano wɔ ɔyarefo 70 (80 ml) mu nyinaa sua. Ayarefo ntam heterogeneity a wɔhwɛ so bere a wɔreyɛ nneɛma atitiriw no nhwehwɛmu (sɛ nhwɛso no, wɔde fã bi a ɛho nhia nhwehwɛmu di dwuma) kyerɛ nsakrae a ɛkɔ so daa wɔ nkwammoaa ahorow ntam, na wɔaboaboa nkwammoaa ahorow ne/anaasɛ microenvironment no ano pefee sɛnea metabolite profile te (Mfonini 2F ). Nhwehwɛmu a wɔyɛe wɔ metabolites biako ho no sii saa nkɛntɛnso ahorow yi so dua na ɛdaa nsonsonoe kɛse adi wɔ nkwammoaa ahorow ne microenvironment ntam. Ɛfata sɛ yɛhyɛ no nsow sɛ nsonsonoe a ɛboro so a wohu ne MNA, a ɛtaa yɛ nea ɛyɛ den wɔ CD45- nkwammoaa ne CD4+ ne CD8+ nkwammoaa a ɛhyɛn akisikuru no mu (Mfonini 3A). Wɔ CD4 + nkwammoaa fam no, saa nkɛntɛnso yi da adi kɛse, na ɛte sɛ nea nneɛma a atwa yɛn ho ahyia no nya MNA a ɛwɔ CD8 + nkwammoaa mu no so nkɛntɛnso kɛse. Nanso, eyi ho nhia, efisɛ ayarefo baanum no mu baasa pɛ na wobetumi asusuw tumor CD8+ nkontaahyɛde ho. MNA akyi no, wɔ nkwammoaa ahorow a ɛwɔ ascites ne tumors mu no, metabolites afoforo a ɛnyɛ nea ɛda adi yiye wɔ TIL mu nso yɛ nea ɛsono emu biara (Mfonini S3 ne S4). Enti, saa nsɛm yi da immunomodulatory metabolites ahorow a ɛhyɛ bɔ adi ma nhwehwɛmu foforo.
(A) MNA a wɔayɛ no sɛnea ɛsɛ wɔ CD4+, CD8+ ne CD45- nkwammoaa a efi ascites ne tumor mu. Adaka mu mfonini no kyerɛ mfinimfini (line), interquartile range (frame hinge) ne data range, a ɛkɔ mmɔho 1.5 interquartile range (frame whisker). Sɛnea wɔaka ho asɛm wɔ Patient Materials and Methods mu no, fa ɔyarefo no limma value no kyerɛ P bo (*P<0.05 ne **P<0.01). (B) MNA nipadua mu nneɛma a ɛsakra ho mfonini (60). Nneɛma a ɛma nipadua no yɛ adwuma: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine ​​a wɔde yɛ nneɛma; NA, nicotinamide a wɔde yɛ nnuru; MNA, 1-methylnicotinamide a wɔde yɛ aduru; 2-PY, 1-methyl- 2-pyridone-5-carboxamide a wɔde yɛ aduru a ɛma obi nya apɔwmuden; 4-PY, 1-methyl-4-pyridone-5-carboxamide a wɔde yɛ aduru a ɛma obi nya apɔwmuden; NR, nicotinamide a wɔde hyɛ nipadua mu; NMN, nicotinamide a wɔde yɛ mononucleotide. Enzyme ahorow (a ɛyɛ ahabammono): NNMT, nicotinamide N-methyltransferase; SIRT, sirtuins, a wɔde wɔn ho hyɛ mu; NAMPT, nicotinamide phosphoribosyl a wɔde fa nneɛma mu; AOX1, aldehyde oxidase 1, a ɛyɛ aduru a ɛma obi nya ahoɔden; NRK, nicotinamide riboside kinase a ɛwɔ nipadua no mu; NMNAT, nicotinamide mono Nukleotide adenylate transferase, a ɛyɛ nea wɔde hyɛ nipadua mu; Pnp1, purine nucleoside phosphorylase a ɛyɛ aduru a ɛma obi nya ahoɔden. (C) t-SNE a scRNA-seq a ascites (grey) ne tumor (kɔkɔɔ; n = ayarefo 3). (D) NNMT nkyerɛkyerɛmu wɔ nkwammoaa dodow ahorow a wɔde scRNA-seq ahu. (E) NNMT ne AOX1 a wɔda no adi wɔ SK-OV-3, nnipa nkwaboaa asaabo (HEK) 293T, T nkwammoaa ne T nkwammoaa a wɔde MNA ayɛ mu. Wɔakyerɛ asɛmfua a wɔabobɔw no sɛ wɔde toto SK-OV-3 ho. Wɔakyerɛ nkyerɛkyerɛmu nhyehyɛe a ɛne SEM (n = 6 apɔwmuden a wɔde ma). Wobu Ct dodow a ɛboro 35 sɛ wontumi nhu (UD). (F) SLC22A1 ne SLC22A2 a wɔda no adi wɔ SK-OV-3, HEK293T, T nkwammoaa ne T nkwammoaa a wɔde 8mM MNA ayɛ ho adwuma mu. Wɔakyerɛ asɛmfua a wɔabobɔw no sɛ wɔde toto SK-OV-3 ho. Wɔakyerɛ nkyerɛkyerɛmu nhyehyɛe a ɛne SEM (n = 6 apɔwmuden a wɔde ma). Wobu Ct dodow a ɛboro 35 sɛ wontumi nhu (UD). (G) Nkwammoaa MNA a ɛwɔ T nkwammoaa a wɔde ma a wɔwɔ apɔwmuden a wɔayɛ adwuma mu wɔ nnɔnhwerew 72 a wɔde MNA ahyɛ mu akyi. Wɔakyerɛ nkyerɛkyerɛmu nhyehyɛe a ɛne SEM (n = 4 apɔwmuden a wɔde ma).
Wɔnam methyl kuw no a wɔde fi S-adenosyl-1-methionine (SAM) mu kɔ nicotinamide (NA) mu denam nicotinamide N-methyltransferase (NNMT; Mfonini 3B) so na ɛyɛ. NNMT da adi boro so wɔ nnipa kokoram ahorow mu na ɛne nkɔanim, ntua ne metastasis wɔ abusuabɔ ( 25-27 ). Sɛnea ɛbɛyɛ na yɛate MNA fibea ase yiye wɔ T nkwammoaa mu wɔ TME mu no, yɛde scRNA-seq dii dwuma de kyerɛɛ NNMT a wɔda no adi wɔ nkwammoaa ahorow nyinaa mu wɔ ascites ne tumors a ɛwɔ HGSC ayarefo baasa mu (Table S5). Nkwammoaa bɛyɛ 6,500 mu nhwehwɛmu kyerɛe sɛ wɔ ascites ne tumor tebea horow mu no, na NNMT a wɔda no adi no yɛ fibroblast ne tumor nkwammoaa dodow a wosusuw sɛ ɛwɔ hɔ no nkutoo (Mfonini 3, C ne D). Ɛfata sɛ yɛhyɛ no nsow sɛ NNMT a ɛda adi pefee biara nni nnipa dodow biara a ɛda PTPRC (CD45 +) adi (Mfonini 3D ne Mfonini S5A), a ɛkyerɛ sɛ wɔde MNA a wɔahu wɔ metabolite spectrum no mu no aba T nkwammoaa mu. Aldehyde oxidase 1 (AOX1) a wɔda no adi no dan MNA ma ɛbɛyɛ 1-methyl-2-pyridone-5-carboxamide (2-PYR) anaa 1-methyl-4-pyridone-5-carboxamide (4- PYR); Mfonini 3B) nso yɛ fibroblasts dodow a ɛda COL1A1 adi (Mfonini S5A) nkutoo, a sɛ wɔka bom a, ɛkyerɛ sɛ T nkwammoaa nni tumi a MNA a wɔtaa yɛ no yɛ adwuma. Wɔde nkwammoaa a ɛto so abien a ɛde ne ho a efi ascites a efi HGSC ayarefo mu (Mfonini S5B; n = 6) (16) dii dwuma de dii saa awosu mu nkwaadɔm a ɛfa MNA ho yi nkyerɛkyerɛmu nhyehyɛe no ho adanse. Bio nso, quantitative polymerase chain reaction (qPCR) nhwehwɛmu a wɔyɛe wɔ donor T nkwammoaa a wɔwɔ apɔwmuden a wɔde MNA saa wɔn yare ho no kyerɛe sɛ sɛ wɔde toto control SK-OV-3 ovarian tumor cells ho a, ɛkame ayɛ sɛ wɔankyerɛ NNMT anaa AOX1 (Mfonini 3E). Saa nneɛma a wɔnhwɛ kwan yi kyerɛ sɛ ebia MNA fi fibroblasts anaa akisikuru mu kɔ T nkwammoaa a ɛbɛn ho wɔ TME mu.
Ɛwom sɛ wɔn a wɔpɛ sɛ wɔpaw wɔn no bi ne abusua a ɛyɛ organic cation transporters 1 kosi 3 (OCT1, OCT2 ne OCT3) a soluble carrier 22 (SLC22) abusua (SLC22A1, SLC22A2 ne SLC22A3) na ɛkyerɛw no de, nanso wɔda so ara nkyerɛkyerɛ nneɛma a ebetumi de MNA mu no mu (28) . QPCR a mRNA a efi apɔwmuden donor T nkwammoaa mu kyerɛɛ SLC22A1 a wɔda no adi a ɛba fam nanso SLC22A2 dodow a wontumi nhu, a ɛsii so dua sɛ wɔadi kan abɔ ho amanneɛ wɔ nhoma ahorow mu (Mfonini 3F) (29). Nea ɛne eyi bɔ abira no, SK-OV-3 ovarian tumor cell line no daa transporters abien no nyinaa dodow a ɛkɔ soro adi (Mfonini 3F).
Sɛnea ɛbɛyɛ na wɔasɔ ahwɛ sɛ ebia T nkwammoaa tumi twetwe amannɔne MNA no, wɔde T nkwammoaa a wɔde ma a wɔwɔ apɔwmuden no yɛɛ aduru nnɔnhwerew 72 wɔ MNA dodow a ɛsono emu biara anim. Sɛ exogenous MNA nni hɔ a, wontumi nhu nkwammoaa mu nneɛma a ɛwɔ MNA mu (Mfonini 3G). Nanso, T nkwammoaa a wɔayɛ adwuma a wɔde exogenous MNA saa wɔn yare no daa nkɔanim a egyina dodow a wɔde ma so adi wɔ MNA a ɛwɔ nkwammoaa no mu no mu, koduu 6 mM MNA (Mfonini 3G). Nea afi mu aba yi kyerɛ sɛ ɛmfa ho sɛ transporter a wɔda no adi no sua na enzyme titiriw a ɛma MNA yɛ adwuma wɔ nkwammoaa mu no nni hɔ no, TIL da so ara tumi fa MNA.
Nneɛma a ɛma nipadua no yɛ adwuma wɔ ayarefo T nkwammoaa mu ne MNA a wɔtwetwe ho sɔhwɛ ahorow a wɔyɛ wɔ vitro no ma hokwan a ɛwɔ hɔ sɛ fibroblasts a ɛne kokoram wɔ abusuabɔ (CAF) no yi MNA na akisikuru nkwammoaa betumi ahwɛ TIL no phenotype ne ne dwumadi so no yɛ kɛse. Sɛnea ɛbɛyɛ na wɔahu nkɛntɛnso a MNA nya wɔ T nkwammoaa so no, wɔmaa T nkwammoaa a wɔde ma a wɔwɔ apɔwmuden no yɛɛ adwuma wɔ vitro bere a MNA wɔ hɔ anaasɛ enni hɔ no, na wɔhwɛɛ sɛnea ɛdɔɔso ne cytokine a wɔyɛ no. Bere a wɔde MNA kaa ho nnafua 7 akyi wɔ dodow a ɛsen biara mu no, nnipa dodow a ɛdɔɔso mmɔho abien no so tew kakra, bere a wɔkɔɔ so kuraa ahoɔden mu wɔ dodow a wɔde ma no nyinaa mu (Mfonini 4A). Bio nso, exogenous MNA ayaresa maa CD4 + ne CD8 + T nkwammoaa a ɛda tumor necrosis factor-α (TNFα; Mfonini 4B) adi no dodow kɔɔ soro. Nea ɛne eyi bɔ abira no, IFN-γ a wɔyɛ wɔ nkwammoaa mu no so tew kɛse wɔ CD4 + T nkwammoaa mu, nanso ɛnyɛ CD8 + T nkwammoaa mu, na nsakrae titiriw biara amma interleukin 2 mu (IL-2; Mfonini 4, C ne D). Enti, enzyme-linked immunosorbent assay (ELISA) a wɔde yɛɛ supernatants a efi saa MNA-treated T cell cultures yi mu no daa nkɔanim kɛse adi wɔ TNFα mu, IFN-γ so tew, na nsakrae biara amma wɔ IL-2 mu (Mfonini 4, E kosi G). . IFN-γ a ɛso tew no kyerɛ sɛ ebia MNA di dwuma wɔ T nkwammoaa no dwumadi a ɛko tia akisikuru no a wosiw ano no mu. Sɛnea ɛbɛyɛ na wɔasuasua nkɛntɛnso a MNA nya wɔ T nkwammoaa-ntamgyinafo cytotoxicity so no, chimeric antigen receptor T (FRα-CAR-T) nkwammoaa a ɛde wɔn ani si folate receptor α ne CAR-T (GFP) a green fluorescent protein (GFP) -CAR-T) nkwammoaa na ɛhwɛ so no yɛ nea wɔde donor peripheral blood mononuclear cells (PBMC) a ɛwɔ apɔwmuden na ɛyɛ. Wɔde CAR-T nkwammoaa yɛɛ aduru nnɔnhwerew 24 wɔ MNA anim, na afei wɔde nnipa SK-OV-3 ovarian tumor nkwammoaa a ɛda folate receptor α adi wɔ effector ne target ratio a ɛyɛ 10:1 mu boom yɛɛ no. MNA ayaresa maa FRα-CAR-T nkwammoaa a wokum no so tew kɛse, a na ɛte sɛ FRα-CAR-T nkwammoaa a wɔde adenosine saa wɔn yare no (Mfonini 4H).
(A) Total viable cell count and population doubling (PD) directly from culture on day 7. Bar graph no gyina hɔ ma nnipa baanum a wɔwɔ apɔwmuden a wɔde mmoa mae no mfinimfini + SEM. Egyina hɔ ma data a efi anyɛ yiye koraa no n = 3 a wɔde wɔn ho sɔhwɛ ahorow mu. (B to D) Wɔde CD3/CD28 ne IL-2 dii dwuma de maa T nkwammoaa yɛɛ adwuma wɔ wɔn MNA dodow mu nnafua 7. Ansa na wɔreyɛ nhwehwɛmu no, wɔde PMA/ionomycin a GolgiStop ka ho kanyan nkwammoaa no nnɔnhwerew 4. TNFα (B) a wɔda no adi wɔ T nkwammoaa mu. Nhwɛso mfonini (benkum so) ne tabular data (nifa so) a ɛkyerɛ TNFα a wɔda no adi wɔ nkwammoaa a nkwa wom mu. IFN-γ (C) ne IL-2 (D) a wɔda no adi wɔ T nkwammoaa mu. Wɔnam flow cytometry so susuw cytokines a wɔda no adi no. Bar graph no gyina hɔ ma mfinimfini (n = 6 a wɔwɔ apɔwmuden a wɔde mmoa ma) + SEM. Fa ɔkwan baako so nhwehwɛmu a ɛfa nsakraeɛ ne susudua a wɔsan yɛ (*P<0.05 ne **P<0.01) di dwuma de kyerɛ P boɔ. Egyina hɔ ma data a efi anyɛ yiye koraa no n = 3 a wɔde wɔn ho sɔhwɛ ahorow mu. (E kosi G) Wɔde CD3/CD28 ne IL-2 dii dwuma de maa T nkwammoaa yɛɛ adwuma wɔ wɔn MNA dodow mu nnafua 7. Wɔboaboaa aduru no ano ansa na wɔreyɛ PMA/ionomycin nkanyan nnɔnhwerew 4 ne akyi. Wɔde ELISA susuw TNFα (E), IFN-γ (F) ne IL-2 (G) dodow. Bar graph no gyina hɔ ma mfinimfini (n = 5 a wɔwɔ apɔwmuden a wɔde mmoa ma) + SEM. P bo a wɔde ɔkwan biako so nhwehwɛmu a ɛfa nsakrae ne susuw a wɔsan susuw (*P<0.05) na ɛkyerɛe. Ntrɛwmu a ɛwɔ nsensanee no kyerɛ anohyeto a ɛwɔ detection no mu. (H) Nkwammoaa mu lysis nhwehwɛmu. Wɔde adenosine (250μM) anaa MNA (10 mM) yɛɛ nsakrae wɔ FRα-CAR-T anaa GFP-CAR-T nkwammoaa mu nnɔnhwerew 24, anaasɛ wogyaw no a wɔansa (Ctrl). Wɔsusuw ɔha biara mu nkyem a wokunkum SK-OV-3 nkwammoaa no. P bo a wɔde Welch t sɔhwɛ (* P <0.5 ne **P <0.01) na ɛkyerɛ.
Sɛnea ɛbɛyɛ a wobenya mfiridwuma mu ntease wɔ MNA-gyina TNFα nkyerɛkyerɛmu nhyehyɛe ho no, wɔhwehwɛɛ nsakrae a ɛba TNFα mRNA mu wɔ T nkwammoaa a wɔde MNA ayɛ wɔn no mu (Mfonini 5A). T nkwammoaa a wɔde ma a wɔwɔ apɔwmuden a wɔde MNA saa wɔn yare no daa TNFα nkyerɛwee dodow a ɛkɔ soro mpɛn abien adi, na ɛkyerɛ sɛ MNA gyina TNFα nkyerɛwee nhyehyɛe so. Sɛnea ɛbɛyɛ na wɔahwehwɛ saa nhyehyɛe a ebetumi aba yi mu no, wɔhwehwɛɛ nkyerɛwee mu nneɛma abien a wonim a ɛhwɛ TNFα so, a ɛne activated T cell nuclear factor (NFAT) ne specific protein 1 (Sp1), mu de buaa MNA a ɛkyekyere proximal TNFα promoter ( 30 ). TNFα promoter no kura mmeae 6 a wɔakyerɛ sɛ NFAT kyekyere ne mmeae 2 a ɛkyekyere Sp1, a ɛka bom wɔ beae biako [-55 base pairs (bp) fi 5’cap] (30). Chromatin immunoprecipitation (ChIP) kyerɛe sɛ sɛ wɔde MNA sa a, Sp1 a ɛkyekyere TNFα promoter no kɔɔ soro mprɛnsa. NFAT a wɔde kaa ho no nso kɔɔ soro na ɛbɛn hia a ɛho hia (Mfonini 5B). Saa nsɛm yi kyerɛ sɛ MNA na ɛhwɛ TNFα a wɔda no adi denam Sp1 nkyerɛwee so, na wɔ ɔkwan ketewaa bi so no, ɛhwɛ NFAT a wɔda no adi so.
(A) Sɛ wɔde toto T nkwammoaa a wɔayɛ no amammerɛ a MNA nnim ho a, nsakrae a ɛba wɔ TNFα a wɔda no adi no mpɛn pii wɔ T nkwammoaa a wɔde MNA ayɛ wɔn no mu. Wɔakyerɛ nkyerɛkyerɛmu nhyehyɛe a ɛne SEM (n = 5 apɔwmuden a wɔde ma). Egyina hɔ ma data a efi anyɛ yiye koraa no n = 3 a wɔde wɔn ho sɔhwɛ ahorow mu. (B) Wɔde TNFα promoter a ɛwɔ T nkwammoaa a wɔde 8 mM MNA ayɛ wɔn yare anaasɛ wɔmfa nni dwuma wɔ NFAT ne Sp1 akyi no ne (Ctrl) ne PMA/ionomycin nkanyan boom nnɔnhwerew 4. Wɔde immunoglobulin G (IgG) ne H3 dii dwuma sɛ nnuru a enye ne nea eye a wɔde siw immunoprecipitation ano. ChIP dodow a wɔde kyerɛe no kyerɛe sɛ Sp1 ne NFAT a ɛkyekyere TNFα promoter no kɔɔ soro mpɛn pii bere a wɔde totoo nea wɔde dii dwuma no ho no. Egyina hɔ ma data a efi anyɛ yiye koraa no n = 3 a wɔde wɔn ho sɔhwɛ ahorow mu. P bo a wɔde t-sɔhwɛ ahorow pii na ɛkyerɛ (*** P <0.01). (C) Sɛ wɔde toto HGSC ascites ho a, T nkwammoaa (a ɛnyɛ cytotoxic) daa TNF a wɔda no adi kɛse wɔ akisikuru no mu adi. Kɔla ahorow no gyina hɔ ma ayarefo ahorow. Wɔayɛ nkwammoaa a wɔada no adi no nhwɛsode a wɔanhyɛ da ayɛ no 300 na wɔayɛ no jittered de asiw overdrawing ano (** Padj = 0.0076). (D) MNA a wɔahyɛ ho nyansa sɛ wɔmfa nyɛ awotwaa mu kokoram. Wɔyɛ MNA wɔ akisikuru nkwammoaa mu ne fibroblasts a ɛwɔ TME mu na T nkwammoaa gye. MNA ma Sp1 a ɛkyekyere TNFα promoter no yɛ kɛse, na ɛma TNFα nkyerɛwee ne TNFα cytokine a wɔyɛ no kɔ soro. MNA nso ma IFN-γ so tew. T nkwammoaa dwumadi a wosiw ano no ma tumi a wokum no so tew na ɛma akisikuru nyin ntɛmntɛm.
Sɛnea amanneɛbɔ kyerɛ no, TNFα wɔ tumi a ɛko tia akisikuru ne akisikuru a egyina anim ne akyi, nanso ɛwɔ dwuma a wonim no yiye wɔ awotwaa mu kokoram nyin ne nea ɛkɔ mmeae afoforo no mu ( 31-33 ). Sɛnea amanneɛbɔ kyerɛ no, TNFα dodow a ɛwɔ ascites ne tumor ntini mu wɔ ayarefo a wɔwɔ awotwaa mu kokoram mu no dɔɔso sen nea ɛwɔ ntini a ɛnyɛ hu mu ( 34-36 ). Wɔ adwinnade ho no, TNFα tumi hwɛ mogya mu nkwammoaa fitaa no dwumadi, dwumadi ne ne dɔɔso so, na ɛsakra kokoram nkwammoaa no phenotype ( 37 , 38 ). Nea ɛne nea wɔahu yi hyia no, nsonsonoe a ɛwɔ awosu mu nkwaadɔm a wɔda no adi mu nhwehwɛmu kyerɛe sɛ na TNF kɔɔ soro kɛse wɔ T nkwammoaa a ɛwɔ akisikuru ntini mu bere a wɔde toto ascites ho no (Mfonini 5C). TNF a wɔda no adi no nkɔanim no daa adi wɔ T nkwammoaa dodow a ɛwɔ phenotype a ɛnyɛ cytotoxic nkutoo mu (Mfonini S5A). Sɛ yɛbɛbɔ no mua a, saa nsɛm yi foa adwene a ɛne sɛ MNA wɔ tumi abien a ɛma nipadua no tumi ko tia nyarewa ne nea ɛma akisikuru nya nkɔso wɔ HGSC mu no so.
Fluorescent labeling a egyina flow cytometry so abɛyɛ ɔkwan titiriw a wɔfa so sua TIL metabolism ho ade. Saa nhwehwɛmu ahorow yi ada no adi sɛ sɛ wɔde toto mogya mu nkwammoaa a ɛwɔ akyi anaa T nkwammoaa a efi lymphoid akwaa a ɛto so abien mu ho a, mpɔtorɔ ne nnipa TIL wɔ su a ɛkorɔn sɛ wɔbɛfa glucose ( 4 , 39 ) ne mitochondrial dwumadi a ɛyera nkakrankakra ( 19 , 40 ). Ɛwom sɛ yɛahu nea afi mu aba a ɛte saa ara wɔ nhwehwɛmu yi mu de, nanso nkɔso titiriw ne sɛ yɛde akisikuru nkwammoaa ne TIL a efi akisikuru ntini koro no ara a wɔatwa mu no mu nneɛma a ɛsakra no bɛtoto ho. Nea ɛne amanneɛbɔ ahorow a atwam yi bi hyia no, akisikuru (CD45-EpCAM +) nkwammoaa a efi ascites ne akisikuru mu no nya glucose a ɛkɔ soro sen CD8 + ne CD4 + T nkwammoaa, na ɛfoa so sɛ wobetumi de glucose a ɛdɔɔso a akisikuru nkwammoaa no gye no atoto T nkwammoaa ho. Adwene a ɛne sɛ T nkwammoaa akansi. TME na ɛyɛ. Nanso, mitochondrial dwumadi a ɛwɔ akisikuru nkwammoaa mu no korɔn sen CD8 + T nkwammoaa no de, nanso mitochondrial dwumadi te sɛ CD4 + T nkwammoaa no de. Saa nsunsuansoɔ yi hyɛ asɛmti a ɛrepue a ɛne sɛ oxidative metabolism ho hia ma tumor cells no mu den ( 41 , 42 ). Wɔsan nso kyerɛ sɛ CD8 + T nkwammoaa betumi ayɛ mmerɛw sɛ oxidative dysfunction bɛba sen CD4 + T nkwammoaa, anaasɛ CD4 + T nkwammoaa betumi de carbon fibea a ɛnyɛ glucose adi dwuma de akura mitochondrial dwumadi mu ( 43 , 44 ). Ɛsɛ sɛ yɛhyɛ no nsow sɛ yɛanhu nsonsonoe biara wɔ glucose a wɔfa anaa mitochondrial dwumadi mu wɔ CD4 + T effectors, T effector memory ne T central memory cells a ɛwɔ ascites mu no ntam. Saa ara nso na nsonsonoe tebea a CD8 + T nkwammoaa a ɛwɔ akisikuru mu no ne nsakrae a ɛba wɔ glucose a wɔfa mu no nni hwee yɛ, na esi nsonsonoe kɛse a ɛda T nkwammoaa a wɔayɛ no amammerɛ wɔ vitro ne nnipa TIL wɔ vivo ntam no so dua ( 22 ). Wɔnam nkwammoaa dodow a wɔkyekyɛ a ɛnyɛ animhwɛ a wɔde dii dwuma so nso sii so dua, na ɛdaa no adi bio sɛ ​​CD45 + / CD3- / CD4 + / CD45RO + nkwammoaa a glucose a wɔfa ne mitochondrial dwumadi dɔɔso sen akisikuru nkwammoaa no abu so nanso ɛwɔ Metabolic active cell population. Saa nnipa dodow yi betumi agyina hɔ ama nnipa kakraa bi a wosusuw sɛ ɛyɛ myeloid suppressor nkwammoaa anaa plasmacytoid dendritic nkwammoaa a wɔahu wɔ scRNA-seq nhwehwɛmu no mu no. Ɛwom sɛ wɔabɔ eyinom abien nyinaa ho amanneɛ wɔ nnipa awotwaa mu akisikuru mu [45] de, nanso ɛda so ara hia Adwuma foforo ne sɛ wɔbɛkyerɛkyerɛ myeloid subpopulation yi mu.
Ɛwom sɛ akwan a egyina flow cytometry so betumi ama nsonsonoe a ɛwɔ glucose ne oxidative metabolism mu wɔ nkwammoaa ahorow ntam no mu ada hɔ de, nanso wonnya nhuu metabolites pɔtee a glucose anaa carbon fibea afoforo yɛ ma mitochondrial metabolism wɔ TME mu. Sɛ wɔde metabolites a ɛwɔ hɔ anaasɛ enni hɔ bɛma TIL kuw ketewaa bi a wɔde ama no a, ɛhwehwɛ sɛ wɔtew nkwammoaa dodow no ho fi ntini a wɔatwa no mu. Enti, yɛn nkwammoaa enrichment kwan a wɔde aka mass spectrometry ho no betumi ama yɛanya nhumu wɔ metabolites a ɛsono sɛnea ɛyɛ kɛse wɔ T nkwammoaa ne tumor nkwammoaa dodow mu wɔ ayarefo nhwɛsode a ɛne ne ho hyia mu. Ɛwom sɛ saa kwan yi wɔ mfaso horow sen fluorescence-activated cell sorting de, nanso metabolite nhomakorabea ahorow bi betumi anya nkɛntɛnso esiane awosu mu pintinn ne / anaasɛ ntɛmntɛm turnover rate ( 22 ). Ne nyinaa mu no, yɛn kwan no tumi huu nneɛma abien a wogye tom a ɛma nipadua no tumi ko tia nyarewa, adenosine ne kynurenine, efisɛ ɛsono kɛse wɔ nhwɛsode ahorow no mu.
Yɛn metabolite nhwehwɛmu a yɛyɛ wɔ akisikuru ne TIL subtypes ho no ma yenya nhumu pii wɔ dwuma a metabolites di wɔ ovarian TME mu no ho. Nea edi kan no, yɛde flow cytometry dii dwuma de hui sɛ nsonsonoe biara nni mitochondrial dwumadi mu wɔ akisikuru ne CD4 + T nkwammoaa ntam. Nanso, LC-MS/MS nhwehwɛmu daa nsakrae kɛse adi wɔ nneɛma a ɛma nipadua no yɛ adwuma pii mu wɔ saa nnipa dodow yi mu, na ɛkyerɛ sɛ nsɛm a wɔde ba awiei wɔ TIL nipadua mu nneɛma a ɛsakra ne ne nipadua mu nneɛma a ɛsakra no nyinaa ho no hwehwɛ sɛ wɔkyerɛ ase yiye. Nea ɛto so abien no, MNA ne metabolite a nsonsonoe kɛse wɔ CD45-nkwaboaa ne T nkwammoaa ntam wɔ ascites mu, na ɛnyɛ akisikuru. Enti, compartmentalization ne tumor beae betumi anya nkɛntɛnso soronko wɔ TIL metabolism so, a ɛtwe adwene si heterogeneity a ebetumi aba wɔ microenvironment bi a wɔde ama mu. Nea ɛto so abiɛsa no, enzyme a ɛma MNA NNMT a wɔda no adi no yɛ CAF nkutoo, a ɛyɛ akisikuru nkwammoaa wɔ ɔkwan ketewaa bi so, nanso wohu MNA dodow a wotumi hu wɔ T nkwammoaa a wonya fi akisikuru mu no mu. NNMT a wɔda no adi boro so wɔ ovarian CAF mu no wɔ tumi a wonim sɛ ɛma kokoram nya nkɔso, a ne fã bi fi CAF metabolism, tumor invasion ne metastasis a wɔhyɛ ho nkuran ( 27 ). Ɛwom sɛ TIL dodow nyinaa yɛ nea ɛkɔ fam de, nanso NNMT a wɔda no adi wɔ CAF mu no ne Cancer Genome Atlas (TCGA) mesenchymal subtype no wɔ abusuabɔ kɛse, a ɛne nkɔmhyɛ a enye wɔ abusuabɔ ( 27 , 46 , 47 ). Awiei koraa no, enzyme AOX1 a ɛma MNA sɛe no nso da adi wɔ CAF nnipa dodow no ara mu, na ɛkyerɛ sɛ T nkwammoaa nni tumi a wɔde sakra MNA no. Nea afi mu aba yi foa adwene a ɛne sɛ ɛwom sɛ adwuma foforo ho hia na ama wɔahu saa ade yi de, nanso MNA dodow a ɛkɔ soro wɔ T nkwammoaa mu no betumi akyerɛ sɛ CAF microenvironment a ɛmma nipadua no tumi ko tia nyarewa wɔ hɔ.
Esiane sɛ MNA a wɔde fa nneɛma no da adi kakraa bi ne protein atitiriw a ɛka MNA nipadua mu nneɛma a ɛsakra ho no dodow a wontumi nhu nti, MNA a ɛwɔ T nkwammoaa mu no yɛ nea wɔnhwɛ kwan. Wɔantumi anhu NNMT anaa AOX1 biara denam scRNA-seq nhwehwɛmu ne qPCR a wɔde wɔn ani asi so a ɛfa akuw abien a wɔde wɔn ho no so. Saa nsunsuansoɔ yi kyerɛ sɛ ɛnyɛ T nkwammoaa na ɛyɛ MNA, na mmom ɛtwe firi TME a atwa ho ahyia no mu. Nsɔhwɛ ahorow a wɔyɛ wɔ vitro mu kyerɛ sɛ T nkwammoaa taa boaboa MNA a efi akyi no ano.
Yɛn in vitro nhwehwɛmu ahorow no ada no adi sɛ exogenous MNA ma TNFα da adi wɔ T nkwammoaa mu na ɛma Sp1 a ɛkyekyere TNFα promoter no yɛ kɛse. Ɛwom sɛ TNFα wɔ dwumadi ahorow a ɛko tia akisikuru ne nea ɛko tia akisikuru de, nanso wɔ awotwaa mu kokoram mu no, TNFα tumi ma awotwaa mu kokoram nyin ( 31-33 ). TNFα a wɔma ɛyɛ neutralization wɔ ovarian tumor cell culture mu anaasɛ TNFα sɛnkyerɛnne a woyi fi hɔ wɔ mouse models mu no betumi ama TNFα-mediated inflammatory cytokine production atu mpɔn na asiw tumor nyin ano ( 32 , 35 ). Enti, wɔ eyi mu no, MNA a wonya fi TME mu no betumi ayɛ adwuma sɛ pro-inflammatory metabolite denam TNFα-dependent mechanism so denam autocrine loop no so, na ɛnam so ama ovarian kokoram a ɛba na ɛtrɛw ( 31 ). Wogyina saa a ebetumi aba yi so reyɛ TNFα blockade ho ade sɛ ade a ebetumi ayɛ aduru a wɔde sa awotwaa mu kokoram ( 37 , 48 , 49 ). Afei nso, MNA sɛe CAR-T nkwammoaa no awuduru a ɛwɔ awotwaa mu akisikuru nkwammoaa mu no, na ɛma wonya adanse foforo a ɛkyerɛ sɛ MNA na ɛma nipadua no tumi ko tia nyarewa. Sɛ wɔka ne nyinaa bom a, nea afi mu aba yi kyerɛ sɛ wɔyɛ nhwɛso bi a ɛma akisikuru ne CAF nkwammoaa yi MNA kɔ TME a ɛwɔ nkwammoaa no akyi mu. Ɛnam (i) TNF-a ɛma ovarian kokoram nyin nkanyan ne (ii) MNA-a ɛma T cell cytotoxic activity inhibition so no, eyi betumi anya tumor nkɛntɛnso abien (Mfonini 5D).
Sɛ yɛde rewie a, ɛdenam nkwammoaa a wɔma ɛyɛ kɛse ntɛmntɛm, nkwammoaa biako a wɔde di dwuma nnidiso nnidiso ne nipadua mu nneɛma a ɛsakra no a wɔaka abom so no, nhwehwɛmu yi daa nsonsonoe kɛse a ɛda akisikuru ne ascites nkwammoaa ntam wɔ immunometabolomic mu adi wɔ HGSC ayarefo mu. Saa nhwehwɛmu a ɛkɔ akyiri yi kyerɛe sɛ nsonsonoe wɔ glucose a wɔfa ne mitochondrial dwumadi mu wɔ T nkwammoaa ntam, na ɛkyerɛe sɛ MNA yɛ nea ɛnyɛ nkwammoaa a ɛyɛ ne ho a ɛhwɛ nipadua no mu tumi a ɛko tia nyarewa so. Saa nsɛm yi nya nkɛntɛnso wɔ sɛnea TME ka T nkwammoaa a ɛma nipadua no yɛ adwuma wɔ nnipa kokoram mu no so. Ɛwom sɛ wɔabɔ akansi tẽẽ a ɛda T nkwammoaa ne kokoram nkwammoaa ntam wɔ aduannuru ho amanneɛ de, nanso nneɛma a ɛsakra no nso betumi ayɛ adwuma sɛ nneɛma a ɛhwɛ nneɛma so a ɛnteɛ de ama akisikuru no akɔ so na ebia asiw nipadua no mu tumi a ɛko tia nyarewa no ano. Nkyerɛkyerɛmu foforo a ɛfa dwuma a saa nneɛma a ɛma nipadua no yɛ adwuma a ɛko tia akisikuru yi di ho no betumi abue akwan foforo a wɔbɛfa so ama nipadua no mu tumi a ɛko tia akisikuru no ayɛ kɛse.
Wɔnam BC kokoram akisikuru ntini akorae a Canadian Tissue Repository Network agye atom no so nyaa ayarefo nhwɛsode ne ayaresabea ho nsɛm. Sɛnea nhyehyɛe a BC Kokoram Nhwehwɛmu Abrabɔ Pa Boayikuw ne British Columbia Sukuupɔn (H07-00463) apene so kyerɛ no, ayarefo no nyinaa nhwɛsode ne ayaresabea ho nsɛm a wonyae nyinaa a wɔkyerɛw penee so anaasɛ wogyaee wɔn pene wɔ ɔkwan a ɛfata so. Wɔde nhwɛsodeɛ no sie BioBank (BRC-00290) a wɔagye atom no mu. Wɔakyerɛ ayarefo su a ɛkɔ akyiri wɔ Tables S1 ne S5 mu. Wɔ cryopreservation mu no, wɔde scalpel di dwuma de mfiri porɔw ɔyarefo no akisikuru nhwɛsode no na afei wɔpia no fa 100-micron filter mu ma wonya nkwammoaa biako a wɔde ahyɛ mu. Wɔde ɔyarefo no ascites no centrifuged wɔ 1500 rpm simma 10 wɔ 4°C de yɛɛ nkwammoaa no pellet na woyii nsu a ɛwɔ so no fii mu. Wɔde nkwammoaa a wonya fii akisikuru ne ascites mu no guu cryopreserved mu wɔ 50% a wɔde ɔhyew ayɛ adwuma wɔ nnipa AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) ne 10% dimethyl sulfoxide mu. Wɔde nkwammoaa biako a wɔakora so a wɔde ahyɛ mu yi yɛɛ nwini na wɔde yɛɛ metabolomics ne metabolite determination a wɔaka ho asɛm wɔ ase hɔ no.
Nsuo a ɛdi mũ no yɛ 0.22 μm a wɔayiyi mu 50:50 a wɔde ahyɛ mu RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) a wɔde nnipa AB serum a ɔhyew a ɛnyɛ adwuma 10% (Sigma-Aldrich), Hepes 12.5 mM (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x aka ho Penicillin Streptomycin (PenStrep) aduru (Thermo Fisher Scientific) ne 50 μMB-mercaptoethanol. Wɔde Hepes 20 mM (Thermo Fisher Scientific) ne 2 mM l-glutamine (Thermo Fisher Scientific) ka AimV (Invitrogen) ho. Na flow cytometer staining buffer no yɛ 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) a wɔde 3% ɔhyew-inactivated AB human serum (Sigma) aka ho. Nkwammoaa mu ahonyade buffer no yɛ 0.22μm PBS a wɔayiyi mu na wɔde 0.5% ɔhyew-inactivated human AB serum (Sigma-Aldrich) ka ho.
Wɔ 37°C a edi mũ mu no, wɔde 10 nM MT DR ne 100 μM 2-NBDG yɛɛ nkwammoaa no ho fĩ simma 30. Afei, wɔde viability dye eF506 yɛɛ nkwammoaa no ho fĩ wɔ 4°C simma 15. San fa nkwammoaa no gu FC Block (eBioscience) ne Brilliant Stain Buffer (BD Biosciences) mu, fa flow cytometer staining buffer (sɛnea nea ɔyɛe no akwankyerɛ kyerɛ no), na fa simma 10 to dan mu hyew mu. Fa nnuru a ɛko tia nyarewa (Table S2) a ɛwɔ flow cytometry staining buffer mu wɔ 4°C mu simma 20 na ɛyɛ nkwammoaa no ho fĩ. San fa nkwammoaa no gu flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R nhyehyɛe) mu ansa na wɔayɛ nhwehwɛmu. Fa SpectroFlo ne FlowJo V10 hwehwɛ cell count data mu, na fa GraphPad Prism 8 yɛ data no. Wɔyɛɛ 2-NBDG ne MT DR no mfinimfini fluorescence ahoɔden (MFI) no log-normalized, na afei wɔde paired t test dii dwuma de yɛɛ akontaabu nhwehwɛmu de buu ayarefo a wɔne wɔn ho hyia no ho akontaa. Yi nnipa dodow a nsɛm a esisii nnu 40 nyinaa fi nhwehwɛmu no mu; hyɛ MFI botaeɛ a ɛyɛ 1 ma botaeɛ bɔne biara ansa na woayɛ akontabuo nhwehwɛmu ne data mfoniniyɛ.
Sɛnea ɛbɛyɛ a yɛde nsaano gating nhyehyɛe a ɛwɔ atifi hɔ nhyehyɛe panel no bɛka ho no, yɛde nkyerɛkyerɛmu a edi mũ a ɛnam nsusuwii anohyeto dua (FAUST) (21) so no dii dwuma de nkwammoaa no maa nnipa dodow no ara bere a yɛayi nkwammoaa a awuwu afi hɔ wɔ FlowJo mu akyi. Yɛde nsa di nea efi mu ba no ho dwuma de ka nnipa dodow a ɛte sɛ nea wɔakyekyɛ wɔn wɔ ɔkwan a ɛnteɛ so (a ɛka PD1 + ne PD1-akisikuru nkwammoaa bom) ne nnipa dodow a wɔakora so bom. Sɛ wɔkyekyem pɛpɛɛpɛ a, nkwammoaa bɛboro 2% na ɛwɔ nhwɛsode biara mu, ma ne nyinaa yɛ nnipa 11.
Wɔde Ficoll gradient density centrifugation dii dwuma de yii PBMC fii nneɛma a wɔde yiyi mogya mu nkwammoaa mu (STEMCELL Technologies) mu. Wɔde CD8 MicroBeads (Miltenyi) yii CD8 + T nkwammoaa fii PBMC mu na wɔtrɛw mu wɔ nsu a edi mũ mu denam TransAct (Miltenyi) so adapɛn 2 sɛnea nea ɔyɛe no akwankyerɛ kyerɛ no. Wɔmaa nkwammoaa no gyinaa hɔ nnafua 5 wɔ nsu a edi mũ a IL-7 (10 ng/ml; PeproTech) wom mu, na afei wɔde TransAct san kanyan wɔn. Da a ɛto so 7 no, sɛnea nea ɔyɛe no akwankyerɛ kyerɛ no, wɔde nnipa CD45 MicroBeads (Miltenyi) dii dwuma de maa nkwammoaa yɛɛ kɛse wɔ mprɛnsa a ɛtoatoa so. Wɔkyekyɛɛ nkwammoaa no mu maa flow cytometry nhwehwɛmu (sɛnea yɛaka ho asɛm wɔ atifi hɔ no), na wɔkyekyɛɛ nkwammoaa ɔpepem biako mu mprɛnsa maa LC-MS/MS nhwehwɛmu. Wɔde LC-MS/MS yɛɛ nhwɛsode ahorow no ho adwuma sɛnea wɔaka ho asɛm wɔ ase ha no. Yɛde ion dodow a ɛyɛ 1,000 buu akontaa sɛ metabolite bo a ayera no. Wɔde ion nɔma nyinaa (TIC) na ɛyɛ nhwɛsode biara normalized, logarithmically converted na automatically normalized wɔ MetaboAnalystR ansa na wɔayɛ nhwehwɛmu.
Wɔde ɔyarefo biara nkwammoaa biako a wɔde ahyɛ mu no yɛɛ nwini na wɔde 40 μm afiri a wɔde yiyi mu no yii mu kɔɔ nsu a edi mũ mu (sɛnea yɛaka ho asɛm wɔ atifi hɔ no). Sɛnea nea ɔyɛe no nhyehyɛe kyerɛ no, wɔde paw pa abiɛsa a ɛtoatoa so denam magnetic bead separation a wɔde MicroBeads (Miltenyi) di dwuma so dii dwuma de maa nhwɛsode ahorow no yɛɛ kɛse maa CD8+, CD4+ ne CD45- nkwammoaa (wɔ nsukyenee so). Ne tiaatwa mu no, wɔsan de nkwammoaa no hyɛ nkwammoaa a ɛma nneɛma yɛ kɛse mu (sɛnea yɛaka ho asɛm wɔ atifi hɔ no) na wɔkan. Wɔde nnipa CD8 nhwiren, nnipa CD4 nhwiren anaa nnipa CD45 nhwiren (Miltenyi) guu nkwammoaa no mu wɔ 4°C simma 15, na afei wɔde cell enrichment buffer hohoroo ho. Wɔde nhwɛsode no fa LS adum (Miltenyi) no mu, na wɔboaboa afã horow a eye ne nea enye ano. Sɛnea ɛbɛyɛ a wɔbɛtew bere tenten no so na wɔama nkwammoaa a wɔsan nya ahoɔden no ayɛ kɛse no, afei wɔde CD8-afã no di dwuma ma CD4+ a wɔde ma no fã a ɛto so abien, na wɔde CD4-afã no di dwuma ma CD45-a wɔde ma a edi hɔ no. Fa ano aduru no sie nsukyenee so wɔ mpaapaemu no nyinaa mu.
Sɛnea ɛbɛyɛ na wɔasiesie nhwɛsode ahorow ama metabolite nhwehwɛmu no, wɔde nkyene a ɛyɛ nwini te sɛ nsukyenee hohoroo nkwammoaa no pɛnkoro, na wɔde methanol 80% 1 ml guu nhwɛsode biara mu, afei wɔde vortex na wɔde yɛɛ nwini ntɛmntɛm wɔ nitrogen nsu mu. Wɔde nhwɛsode ahorow no yɛɛ nwini-thaw kyinhyia abiɛsa na wɔde centrifuged wɔ 14,000 rpm simma 15 wɔ 4°C. Wɔma nsu a ɛwɔ soro a metabolites no wom no yɛ hyew kosi sɛ ɛbɛyow. Wɔsan de metabolites no guu 50 μl 0.03% formic acid mu, de vortex yɛɛ no ​​ma ɛfrafrae, na afei wɔde centrifuged guu mu de yii nneɛma a asɛe no fii mu.
Yi metabolites sɛnea yɛaka ho asɛm wɔ atifi hɔ no. Fa nsu a ɛwɔ soro no gu nsu chromatography toa a ɛyɛ adwuma yiye mu ma yɛyɛ metabolomics nhwehwɛmu. Fa random treatment protocol di dwuma de sa sample biara yare denam nkwammoaa dodow a ɛte saa ara so de siw batch effects ano. Yɛyɛɛ wiase nyinaa metabolites a wɔadi kan atintim wɔ AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50) so no su mu nhwehwɛmu. Wɔde MultiQuant version 2.1 software (Applied Biosystems SCIEX) na ɛyɛɛ chromatographic nhwehwɛmu ne peak area integration.
Wɔde ion dodow a ɛyɛ 1000 dii dwuma de buu metabolite bo a ɛyera no ho akontaa, na wɔde TIC a ɛwɔ nhwɛsode biara mu dii dwuma de buu normalized peak area a ɛwɔ metabolite biara a wɔahu no mu de siesiee nsakrae a ɛnam adwinnade nhwehwɛmu a efi nhwɛsode no ho dwumadie mu baeɛ no so. Bere a wɔayɛ TIC no normalized akyi no, wɔde MetaboAnalystR(51) (default parameter) di dwuma ma logarithmic nsakrae ne automatic norm line scaling. Yɛde PCA a vegan R package ka ho dii dwuma de yɛɛ nhwehwɛmu nhwehwɛmu wɔ metabolome nsonsonoe a ɛwɔ nhwɛsode ahorow ntam, na yɛde partial redundancy nhwehwɛmu yɛɛ ayarefo mu nhwehwɛmu. Fa Ward kwan no yɛ ɔhyew map dendrogram de boaboa Euclidean kwan a ɛda nhwɛsode ahorow ntam no ano. Yɛde limma (52) dii dwuma wɔ standardized metabolite dodow so de kyerɛɛ nsonsonoe a ɛdɔɔso metabolites wɔ nkwammoaa su ne microenvironment nyinaa mu. Sɛnea ɛbɛyɛ a nkyerɛkyerɛmu no bɛyɛ mmerɛw no, yɛde kuw no kyerɛ parameter no di dwuma de kyerɛ nhwɛsode no, na yesusuw nkwammoaa ahorow a ɛwɔ microenvironment no mu sɛ kuw biara (n = akuw 6); ma significance test no, yɛyɛɛ susuw abiɛsa mpɛn pii maa metabolite biara Sɛnea ɛbɛyɛ a yɛbɛkwati atoro nsɛso no, wɔde ɔyarefo no kaa ho sɛ akwanside wɔ limma nhyehyɛe no mu. Sɛnea ɛbɛyɛ a yɛbɛhwɛ nsonsonoe a ɛwɔ metabolites mu wɔ ayarefo ahorow ntam no, yɛyɛɛ nsakrae wɔ limma model a ayarefo ka ho no mu wɔ ɔkwan a ɛyɛ pintinn so. Yɛbɔ amanneɛ sɛ nsonsonoe a wɔadi kan akyerɛ wɔ nkwammoaa su ne microenvironment a ɛwɔ Padj <0.05 (Benjamini-Hochberg nteɛso) ntam no ho hia.
Wɔ ahoɔden a wɔde Miltenyi Dead Cell Removal Kit (>80% viability) dii dwuma akyi no, wɔyɛɛ nkwammoaa biako transcriptome nnidiso nnidiso wɔ ascites a wɔahyɛ no nwini a ɛte ase nyinaa ne akisikuru nhwɛsode ahorow no so denam 10x 5′gene expression protocol so. Wɔyɛɛ nsɛm anum a na akisikuru ne ascites a ɛne ne ho hyia mu nhwehwɛmu, ɛwom sɛ nkwa a ɛba fam a wonya fii akisikuru nhwɛsode biako mu no amma wɔantumi anka ho de. Sɛnea ɛbɛyɛ a yebenya ayarefo a wɔpaw wɔn pii no, yɛkaa ɔyarefo biara nhwɛsode boom wɔ 10x chromium controller no akwan so, na yɛyɛɛ ascites ne tumor mmeae no mu nhwehwɛmu wɔ ɔkwan soronko so. Bere a wɔayɛ no nnidiso nnidiso akyi [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; sɛ wɔkyekyem pɛpɛɛpɛ a, akenkan 73,488 ne 41,378 wɔ nkwammoaa biara mu ma akisikuru ne akisikuru]], yɛde CellSNP ne Vireo (53) dii dwuma (a egyina CellSNP so sɛ The common human SNP (VCF) a GRCh38 de ma no yɛ nea wɔde ma.Yɛde SNPRelate di dwuma de kyerɛ sɛnea ɔyarefo no genotype tebea (IBS) a ɛbɛn sen biara (IBS), excluding unassigned cells and cells identified as duplexes and matching donors between ascites and tumor samples (54) Yɛde nsɛm abiɛsa a nkwammoaa gyina hɔ ma pii wɔ akisikuru no mu ne ascites no siei maa downstream analysis Bere a yɛayɛ mass filtration step wɔ scater (55) ne scran (56) BioConductor packaging no mu akyi (2792 ne 4183 nkwammoaa fi tumor ne ascites, sɛnea ɛte biara) ma nhwehwɛmu Yɛde igraph's (57) Louvain clustering of shared nearest neighbor network (SNN) a egyina Jaccard kwan a ɛkɔ cluster nkwammoaa so denam nkyerɛkyerɛmu so a wɔakyerɛkyerɛ mu denam CD8A ne GZMA a wɔda no adi so, a subclusters a ɛwɔ ribosomal protein nkyerɛkyerɛmu a ɛba fam no nka ho, yenyaa Izar ne ne mfɛfo (16) data a wɔatintim no, a wɔn t-SNE embedding ka ho, betumi adi nkyerɛkyerɛmu a ɛka bom wɔ immune cell markers ne NNMT nkyerɛkyerɛmu no so.
Wɔnam Ficoll gradient density centrifugation so yii PBMC fii nneɛma a wɔde yiyi mogya mu nkwammoaa mu (STEMCELL Technologies) mu. Wɔde CD3 beads (Miltenyi) yii CD3 + nkwammoaa fii PBMC mu. Sɛ MNA wɔ hɔ anaasɛ enni hɔ a, wɔde CD3 a wɔakyekyere no mprɛte (5μg/ml), CD28 a ɛtumi nwura mu (3μg/ml) ne IL-2 (300 U/ml; Proleukin) yɛɛ CD3 + nkwammoaa no adwuma. Da a etwa to a wɔtrɛw mu no, wɔde flow cytometry hwɛɛ sɛnea ebetumi ayɛ adwuma (Fixable Viability Dye eFluor450, eBioscience) ne sɛnea ɛdɔɔso (123count eBeads, Thermo Fisher Scientific). Hwɛ effector dwumadie denam PMA (20 ng/ml) ne ionomycin (1μg/ml) a wode GolgiStop bɛkanyan nkwammoaa no so nnɔnhwereɛ 4, na hwɛ CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) ne TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD) so. Fa PMA (20 ng/ml) ne ionomycin (1μg/ml) kanyan qPCR ne ChIP nkwammoaa no nnɔnhwerew 4. Wɔde PMA (20 ng/ml) ne ionomycin (1 μg/ml) boaboaa ELISA supernatant ano ansa na wɔrekanyan no ne bere a wɔakanyan no akyi nnɔnhwerew 4.
Di nea ɔyɛe no nhyehyɛe akyi de yi RNA fi mu denam RNeasy Plus Mini Kit (QIAGEN) so. Fa QIAshredder (QIAGEN) di dwuma de ma nhwɛsode no yɛ biako. Fa RNA to cDNA kit a ɛwɔ tumi kɛse (Thermo Fisher Scientific) di dwuma de yɛ DNA a ɛka ho (cDNA). Fa TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) di dwuma de kyerɛ awosu mu nkwaadɔm a wɔda no adi (sɛnea nea ɔyɛe no nhyehyɛe kyerɛ) denam nhwehwɛmu ahorow a edidi so yi so: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate a ɛwɔ Hydrogen mu (GAPDH)] ne Hs01010726_m1 (SLC22A2). Wɔde nhwɛsodeɛ no yɛɛ adwuma wɔ StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) so wɔ MicroAmp fast optical 96-well reaction plate (Applied Biosystems) a MicroAmp optical film ka ho. Wobu Ct bo biara a ɛboro 35 sɛ ɛboro detection threshold no so na wɔahyɛ no agyirae sɛ wontumi nhu.
Yɛ ChIP sɛnea wɔadi kan aka ho asɛm (58). Ne tiawa mu no, wɔde formaldehyde (ne dodow a etwa to 1.42%) yɛɛ nkwammoaa no ho adwuma na wɔde too dan mu hyew mu simma 10. Fa supplemented swelling buffer (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl ne 0.1% NP-40) di dwuma wɔ nsukyenee so simma 10, afei san fa gu immunoprecipitation buffer mu sɛnea wɔaka ho asɛm ( 58 ). Afei wɔde nhwɛsode no sonicated ne kyinhyia a edidi so yi: 10 kyinhyia (20 1-second pulses) ne static bere a 40 sikani. Fa ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) ne SP1 (Cell Signaling Technology; 3μl) antibodies ne nhwɛsode no gu 4°CC wosow mu anadwo mũ nyinaa. Fa protein A nhwiren (Thermo Fisher Scientific) ne nhwɛsode no to 4°C a wowosow no brɛoo dɔnhwerew 1, afei fa chelex nhwiren (Bio-Rad) di dwuma ma DNA no yɛ kɛse, na fa proteinase K (Thermo Fisher) di dwuma ma protein no di dwuma. Wɔnam PCR so huu TNFα promoter: anim, GGG TAT CCT TGA TGC TTG TGT; nea ɛne no bɔ abira no, GTG CCA ACA ACT GCC TTT ATA TG (207-bp aduru). Image Lab (Bio-Rad) na ɛyɛɛ mfonini ahorow no na wɔde ImageJ software na ɛkyerɛɛ dodow.
Wɔboaboaa cell culture supernatant no ano sɛnea yɛaka ho asɛm wɔ atifi hɔ no. Wɔyɛɛ gyinaesi no sɛnea nea ɔyɛe no akwan a ɛfa nnipa TNFα ELISA kit (Invitrogen), nnipa IL-2 ELISA kit (Invitrogen) ne nnipa IFN-γ ELISA kit (Abcam) kyerɛ. Sɛnea nea ɔyɛe no nhyehyɛe kyerɛ no, wɔde nsu a ɛwɔ soro no guu mu 1:100 de huu TNFα ne IL-2, ne 1:3 de huu IFN-γ. Fa EnVision 2104 Multilabel Reader (PerkinElmer) susuw sɛnea ɛtwetwe nsu wɔ 450 nm.
Wɔnam Ficoll gradient density centrifugation so yii PBMC fii nneɛma a wɔde yiyi mogya mu nkwammoaa mu (STEMCELL Technologies) mu. Wɔde CD3 beads (Miltenyi) yii CD3 + nkwammoaa fii PBMC mu. Sɛ MNA wɔ hɔ anaasɛ enni hɔ a, wɔde CD3 a wɔakyekyere mprɛte (5μg/ml), CD28 a ɛtumi nwura mu (3μg/ml) ne IL-2 (300 U/ml; Proleukin) yɛɛ CD3 + nkwammoaa no adwuma nnafua 3. Nnafua 3 akyi no, wɔboaboaa nkwammoaa no ano na wɔde nkyene 0.9% hohoroo ho, na wɔde pellet no yɛɛ nwini ntɛm ara. Wɔnam flow cytometry (Cytek Aurora; 3L-16V-14B-8R nhyehyɛe) so yɛɛ nkwammoaa dodow a wɔde 123count eBeads dii dwuma.
Yi metabolites sɛnea yɛaka ho asɛm wɔ atifi hɔ no. Wɔsan yɛɛ aduru a ayow no wɔ dodow a ɛyɛ 4000 cell equivalents/μl. Fa reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) ne CORTECS T3 adum (2.1×150 mm, abɔdeɛ nketenkete kɛseɛ 1.6-μm, pore kɛseɛ 120-Å; #186008500, Waters) hwehwɛ nhwɛsoɔ no mu. Polar mass spectrometer (6470, Agilent), a electrospray ionization yɛ adwuma wɔ ɔkwan pa so. Mobile phase A yɛ 0.1% formic acid (wɔ H2O mu), mobile phase B yɛ 90% acetonitrile, 0.1% formic acid. LC gradient no yɛ simma 0 kɔsi 2 ma 100% A, simma 2 kɔsi 7.1 ma 99% B, ne simma 7.1 kɔsi 8 ma 99% B. Afei san fa mobile phase A no kari pɛ wɔ adum no mu wɔ nsuo a ɛsen 0.6 ml/min mu simma 3. . Nsuo a ɛsen no yɛ 0.4ml/min, na wɔma adum dan no yɛ hyew kodu 50°C. Fa MNA nnuru gyinapɛn a ɛho tew (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) di dwuma de si bere a wɔde sie (RT) ne nsakrae (RT = simma 0.882, nsakrae 1 = 137→94.1, nsakrae 2 = 137→92 , Nsakrae 3 = 137→78) si hɔ. Sɛ nsakraeɛ mmiɛnsa no nyinaa ba wɔ berɛ a ɛfata a wɔde sie mu a, wɔde nsakraeɛ 1 di dwuma de kyerɛ dodoɔ de hwɛ sɛ ɛyɛ pɔtee. Wɔde stock solution (1 mg/ml) a wɔde ahyɛ mu asia nnidiso nnidiso na ɛyɛɛ MNA (Toronto Research Chemical Company) gyinapɛn curve no de nyaa gyinapɛn a ɛyɛ 0.1, 1.0, 10 ne 100 ng/ml ne 1.0 ne 10μg/ml nsuo. Anohyeto a wɔde hu no yɛ 1 ng/ml, na linear mmuae no yɛ 10 ng/ml ne 10μg/ml ntam. Wɔde nhwɛsode ne gyinapɛn microliter abien a wɔde bɛhyɛ mu biara di dwuma de yɛ LC/MS nhwehwɛmu, na wɔde nhwɛsode a wɔde hwɛ sɛnea nneɛma te a wɔadi afra di dwuma wɔ injection awotwe biara mu de hwɛ hu sɛ nhwehwɛmu asɛnka agua no gyina pintinn. Na MNA mmuae a ɛwɔ nkwammoaa nhwɛsode a wɔde MNA ayɛ no nyinaa mu wɔ nhwehwɛmu no linear range mu. Wɔde MassHunter quantitative analysis software (v9.0, Agilent) na ɛyɛɛ data nhwehwɛmu.
Wɔfaa awo ntoatoaso a ɛto so abien αFR-CAR construct no fii Song ne ne mfɛfo hɔ. (59) no. Ne tiawa mu no, construct no kura nsɛm a edidi so yi: CD8a kannifo ntoatoaso, onipa αFR-specific single-chain variable fragment, CD8a hinge ne transmembrane region, CD27 intracellular domain ne CD3z intracellular domain. Wɔnam GenScript so yɛɛ CAR ntoatoaso a edi mũ no, na afei wɔde yɛɛ clone wɔ awo ntoatoaso a ɛto so abien lentiviral expression vector a ɛwɔ GFP expression cassette a wɔde dii dwuma de hwɛɛ transduction efficiency no atifi.
Wɔnam HEK293T nkwammoaa a wɔde hyɛ nipadua mu so na ɛyɛ lentivirus [American Type Culture Collection (ATCC); a wɔadua wɔ Dulbecco’s modified Eagle medium a 10% fetal bovine serum (FBS) ne 1% PenStrep wom mu, na wɔde CAR-GFP vector ne The packaging plasmids (psPAX2 ne pMD2.G, Addgene) de lipofection amine (Sigma-Aldrich) di dwuma. Wɔboaboaa nsu a ɛwɔ soro a mmoawa wom no ano wɔ nnɔnhwerew 48 ne 72 akyi wɔ transfection akyi, yii mu, na wɔde ultracentrifugation yɛɛ no ​​kɛse. Fa mmoawa a ɛwɔ soro a wɔahyɛ no den no sie -80°C kosi sɛ wɔbɛdan no.
Wɔnam Ficoll gradient density centrifugation so yiyi PBMC fi nneɛma a wɔde yiyi mogya mu nkwammoaa a wɔde ma a ɛwɔ apɔwmuden mu (STEMCELL Technologies) mu. Fa positive selection CD8 microbeads (Miltenyi) di dwuma de yi CD8+ nkwammoaa fi PBMC mu. Kanyan T nkwammoaa denam TransAct (Miltenyi) ne TexMACS aduru mu [Miltenyi; wɔde nnipa mogya mu 3% a wɔde ɔhyew ayɛ adwuma, PenStrep 1% ne IL-2 (300 U/ml) aka ho]. Nnɔnhwerew aduonu anan wɔ nkanyan akyi no, wɔde lentivirus (10 μl concentrated virus supernatant wɔ nkwammoaa 106 biara mu) danee T nkwammoaa no. Nnafua 1 kosi 3 akyi wɔ transduction wɔ Cytek Aurora (wɔ FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), hwehwɛ GFP a wɔda no adi wɔ nkwammoaa no mu de kyerɛ sɛ anyɛ yiye koraa no transduction no tumi yɛ 30% .
Wɔde CAR-T nkwammoaa yɛɛ aduru nnɔnhwerew 24 wɔ Immunocult (STEMCELL Technologies; wɔde 1% PenStrep ahyɛ mu) wɔ tebea horow a edidi so yi mu: wɔansa no yare, wɔde 250 μM adenosine anaa 10 mM MNA ayɛ ho adwuma. Bere a wɔadi kan ayɛ ho adwuma akyi no, wɔde PBS hohoroo CAR-T nkwammoaa no na wɔde SK-OV-3 nkwammoaa 20,000 kaa ho [ATCC; wɔ McCoy 5A medium (Sigma-Aldrich) a wɔde 10% FBS ne 1% PenStrep ahyɛ mu wɔ 10 mu: Wɔmaa effector ne target ratio a ɛyɛ 1 no yɛɛ kɛse mprɛnsa wɔ Immunocult medium a wɔde aka ho mu. Wɔde SK-OV-3 nkwammoaa ne SK-OV-3 nkwammoaa a wɔde digitalis saponin (0.5mg/ml; Sigma-Aldrich) ayɛ no dii dwuma sɛ nea enye ne nea ɛyɛ papa. Bere a wɔde nnɔnhwerew 24 boom duaa akyi no, wɔboaboaa nsu a ɛwɔ soro no ano na wɔsusuw lactate dehydrogenase (LDH) no sɛnea nea ɔyɛe no akwankyerɛ kyerɛ (LDH Glo Cytotoxicity Assay Kit, Promega). Wɔde LDH a ɛwɔ soro no guu LDH buffer mu 1:50. Wɔde ɔkwan a edidi so yi na ɛsusuu ɔha biara mu nkyekyɛmu a wɔde kum nnipa: ɔha biara mu nkyekyɛmu a wɔakum = ɔha biara mu nkyekyɛmu a wɔde nteɛso / awudi dodoɔ a ɛkyɛn so x 100%, baabi a nteɛsoɔ ɔha mu nkyekyɛmu = co-culture-T cells nko ara, ne kum dodoɔ a ɛkyɛn soɔ = positive control-negative control.
Sɛnea wɔakyerɛkyerɛ mu wɔ nkyerɛwee anaa nneɛma ne akwan no mu no, fa GraphPad Prism 8, Microsoft Excel anaa R v3.6.0 di dwuma ma akontaabu nhwehwɛmu. Sɛ wɔboaboa nhwɛsode pii ano fi ɔyarefo koro no ara hɔ (te sɛ ascites ne tumor) a, yɛde paired t test di dwuma anaasɛ yɛde ɔyarefo no ka ho sɛ random effect wɔ linear anaa generalized model mu sɛnea ɛfata. Wɔ metabolomics nhwehwɛmu mu no, wɔyɛ hia a ɛho hia sɔhwɛ no mprɛnsa.
Sɛ wopɛ nneɛma foforo a wode bɛka saa asɛm yi ho a, yɛsrɛ wo hwɛ http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
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Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Sacharias), Abigail Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. DeBerardinis), Russell G. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T. 1999, na ɔde ne nsa kyerɛɛ ne so, na ɔde ne nsa kyerɛɛ ne so bio.
MNA boa ma T nkwammoaa no tumi a ɛko tia nyarewa no siw ano na egyina hɔ ma nipadua no mu tumi a ɛko tia nyarewa a wɔde bɛsa nnipa kokoram.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Sacharias), Abigail Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. DeBerardinis), Russell G. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T. 1999, na ɔde ne nsa kyerɛɛ ne so, na ɔde ne nsa kyerɛɛ ne so bio.
MNA boa ma T nkwammoaa no tumi a ɛko tia nyarewa no siw ano na egyina hɔ ma nipadua no mu tumi a ɛko tia nyarewa a wɔde bɛsa nnipa kokoram.
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