ʻO nā metabolites immunomodulatory kahi hiʻohiʻona koʻikoʻi o ka microenvironment tumor (TME), akā me kekahi mau ʻokoʻa, ʻaʻole i ʻike nui ʻia ko lākou ʻano. Maanei, ua kālailai mākou i nā tumors a me nā cell T mai nā tumors a me nā ascites o nā maʻi me ka serous carcinoma kiʻekiʻe (HGSC) e hōʻike i ka metabolome o kēia mau keʻena TME like ʻole. Loaʻa i nā Ascites a me nā cell tumor nā ʻokoʻa metabolite nui. Ke hoʻohālikelike ʻia me nā ascites, ua hoʻonui nui ʻia nā cell T tumor-infiltrating i ka 1-methylnicotinamide (MNA). ʻOiai ua kiʻekiʻe ka pae o MNA i loko o nā cell T, ʻo ka hōʻike ʻana o ka nicotinamide N-methyltransferase (kahi enzyme e hoʻoulu ai i ka hoʻoili ʻana o nā hui methyl mai S-adenosylmethionine i ka nicotinamide) ua kaupalena ʻia i nā fibroblasts a me nā cell tumor. Ma ke ʻano hana, hoʻoulu ka MNA i nā cell T e huna i ka cytokine tumor necrosis factor alpha e hoʻolaha ana i ka tumor. No laila, hāʻawi ka MNA i loaʻa mai TME i ka hoʻoponopono ʻana o ka pale ʻana o nā cell T a hōʻike i kahi pahuhopu immunotherapy hiki ke mālama ʻia i ka maʻi kanesa kanaka.
Hiki i nā metabolites i loaʻa mai ka tumor ke loaʻa ka hopena pale nui i ka pale ʻana i ka tumor, a ʻoi aku ka nui o nā hōʻike e hōʻike ana hiki iā lākou ke lawelawe ma ke ʻano he mana hoʻokele koʻikoʻi no ka holomua o ka maʻi (1). Ma waho aʻe o ka hopena Warburg, ua hoʻomaka ka hana hou e wehewehe i ke kūlana metabolic o nā cell tumor a me kona pilina me ke kūlana pale o ka microenvironment tumor (TME). Ua hōʻike nā haʻawina ma nā hiʻohiʻona ʻiole a me nā cell T kanaka e hiki i ka metabolism glutamine (2), ka metabolism oxidative (3) a me ka metabolism glucose (4) ke hana kūʻokoʻa ma nā hui cell immune like ʻole. He nui nā metabolites i loko o kēia mau ala e pale aku i ka hana anti-tumor o nā cell T. Ua hōʻoia ʻia e hiki i ka blockade o ka coenzyme tetrahydrobiopterin (BH4) ke hōʻino i ka hoʻonui ʻia ʻana o nā cell T, a ʻo ka hoʻonui ʻia o BH4 i loko o ke kino ke hoʻoikaika i ka pane pale anti-tumor i hoʻopili ʻia e CD4 a me CD8. Eia kekahi, hiki ke hoʻopakele ʻia ka hopena immunosuppressive o kynurenine e ka hoʻokele ʻana o BH4 (5). I loko o ka isocitrate dehydrogenase (IDH) mutant glioblastoma, ʻo ka hoʻokuʻu ʻana o ka enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) e kāohi ana i ka hoʻāla ʻana o nā cell T, ka hoʻonui ʻana a me ka hana cytolysis (6). I kēia manawa, ua hōʻike ʻia ua hana ʻia ka methylglyoxal, kahi huahana o ka glycolysis, e nā cell suppressor o ke kumu myeloid, a hiki i ka hoʻoili ʻana o nā cell T o ka methylglyoxal ke kāohi i ka hana effector T cell. I ka mālama ʻana, hiki i ka neutralization o ka methylglyoxal ke lanakila i ka hana o nā cell suppressor i loaʻa mai ka myeloid (MDSC) a hoʻoikaika synergistically i ka checkpoint blockade therapy i nā hiʻohiʻona ʻiole (7). Hoʻoikaika pū kēia mau haʻawina i ke kuleana koʻikoʻi o nā metabolites i loaʻa mai TME i ka hoʻoponopono ʻana i ka hana a me ka hana o nā cell T.
Ua hōʻike nui ʻia ka hana ʻino o nā cell T i loko o ke kanesa ovarian (8). ʻO kēia kekahi hapa ma muli o nā ʻano metabolic i loko o ka hypoxia a me ka vasculature tumor abnormal (9), kahi e hopena ai i ka hoʻololi ʻana o ka glucose a me ka tryptophan i nā huahana e like me ka lactic acid a me ka kynurenine. Hoʻemi ka lactate extracellular nui i ka hana ʻana o interferon-γ (IFN-γ) a hoʻokele i ka hoʻokaʻawale ʻana o nā subgroups myelosuppressive (10, 11). ʻO ka ʻai ʻana o ka tryptophan e kāohi pololei i ka hoʻonui ʻana o nā cell T a kāohi i ka hōʻailona receptor cell T (12-14). Me kēia mau nānā ʻana, ua hana ʻia ka nui o ka hana e pili ana i ka metabolism immune i loko o ka moʻomeheu cell T in vitro me ka hoʻohana ʻana i nā media i hoʻonui ʻia, a i ʻole i kaupalena ʻia i nā hiʻohiʻona ʻiole homologous i vivo, ʻaʻole kekahi o ia mau mea e hōʻike piha ana i ka heterogeneity o nā maʻi kanesa kanaka a me ke kaiapuni macro a me micro physiological.
ʻO kahi hiʻohiʻona maʻamau o ka maʻi ʻaʻai ovarian ka laha peritoneal a me ke ʻano o nā ascites. ʻO ka hōʻiliʻili ʻana o ka wai cell i loko o nā ascites e pili ana me ka maʻi holomua a me ka prognosis maikaʻi ʻole (15). Wahi a nā hōʻike, he hypoxic kēia ʻāpana kū hoʻokahi, he kiʻekiʻe nā pae o ka vascular endothelial growth factor (VEGF) a me ka indoleamine 2,3-dioxygenase (IDO), a ua komo ʻia e nā cell regulatory T a me nā cell inhibitory myeloid (15-18). ʻOkoʻa paha ke kaiapuni metabolic o nā ascites mai ko ka tumor ponoʻī, no laila ʻaʻole maopopo ka reprogramming o nā cell T i ka peritoneal space. Eia kekahi, ʻo nā ʻokoʻa koʻikoʻi a me ka heterogeneity ma waena o nā ascites a me nā metabolites i loaʻa i loko o ke kaiapuni tumor e pale aku i ka komo ʻana o nā cell immune a me kā lākou hana ma nā tumors, a pono ka noiʻi hou aku.
I mea e hoʻoponopono ai i kēia mau pilikia, ua hoʻolālā mākou i kahi ʻano hoʻokaʻawale cell koʻikoʻi a me ka chromatography wai tandem mass spectrometry (LC-MS/MS) e aʻo ai i nā ʻano cell like ʻole (me nā CD4 + a me CD8 + T cell) a me loko a ma waena o nā tumors. Hoʻopili kāna metabolites i nā cell i loko o ka ascites like a me ke kaiapuni tumor o ka mea maʻi. Hoʻohana mākou i kēia ʻano hana me ka cytometry kahe kiʻekiʻe a me ka hoʻonohonoho RNA cell hoʻokahi (scRNA-seq) e hāʻawi i kahi kiʻi i hoʻoholo nui ʻia o ke kūlana metabolic o kēia mau heluna koʻikoʻi. Ua hōʻike kēia ʻano hana i ka piʻi nui ʻana o ka pae o 1-methylnicotinamide (MNA) i loko o nā cell T tumor, a ua hōʻike nā hoʻokolohua in vitro ʻaʻole i ʻike mua ʻia ka hopena immunomodulatory o MNA ma ka hana cell T. Ma keʻano laulā, hōʻike kēia ʻano hana i nā pilina metabolic like ma waena o nā tumors a me nā cell immune, a hāʻawi i nā ʻike kūikawā i nā metabolites hoʻoponopono immune, kahi mea pono paha no ka mālama ʻana i nā manawa lapaʻau immunotherapy ovarian cancer e pili ana i ka cell T.
Ua hoʻohana mākou i ka cytometry kahe kiʻekiʻe-dimensional e helu like i ka lawe ʻana o ka glucose [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) a me ka hana mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) he mau māka maʻamau e hoʻokaʻawale ana i nā cell immune a me nā heluna cell tumor (Papa S2 a me ke Kiʻi S1A). Ua hōʻike kēia loiloi i ka hoʻohālikelike ʻia me nā cell T, ʻoi aku ka kiʻekiʻe o ka lawe ʻana o ka glucose i nā ascites a me nā cell tumor, akā he ʻokoʻa liʻiliʻi i ka hana mitochondrial. ʻO ka awelika o ka lawe ʻana o ka glucose o nā cell tumor [CD45-EpCAM (EpCAM)+] he ʻekolu a ʻehā manawa o nā cell T, a ʻo ka awelika o ka lawe ʻana o ka glucose o nā cell CD4 + T he 1.2 manawa o nā cell CD8 + T, e hōʻike ana he ʻokoʻa nā koi metabolic o nā lymphocytes Tumor infiltrating (TIL) ʻoiai ma ka TME like (Kiʻi 1A). I ka hoʻohālikelike ʻana, ua like ka hana mitochondrial i loko o nā cell tumor me ko nā cell CD4 + T, a ʻoi aku ka kiʻekiʻe o ka hana mitochondrial o nā ʻano cell ʻelua ma mua o nā cell CD8 + T (Kiʻi 1B). Ma ke ʻano laulā, hōʻike kēia mau hopena i ka pae metabolic. ʻOi aku ka kiʻekiʻe o ka hana metabolic o nā cell tumor ma mua o nā cell CD4 + T, a ʻoi aku ka kiʻekiʻe o ka hana metabolic o nā cell CD4 + T ma mua o nā cell CD8 + T. ʻOiai kēia mau hopena ma waena o nā ʻano cell, ʻaʻohe ʻokoʻa mau i ke kūlana metabolic o nā cell CD4 + a me CD8 + T a i ʻole ko lākou mau ʻāpana pili i nā ascites i hoʻohālikelike ʻia me nā tumors (Kiʻi 1C). I ka hoʻohālikelike ʻana, i ka ʻāpana CD45-cell, ua piʻi ka hapa o nā cell EpCAM+ i loko o ka tumor i hoʻohālikelike ʻia me nā ascites (Kiʻi 1D). Ua ʻike pū mākou i kahi ʻokoʻa metabolic maopopo ma waena o nā ʻāpana cell EpCAM+ a me EpCAM-. ʻOi aku ka kiʻekiʻe o ka glucose uptake a me ka hana mitochondrial o nā cell EpCAM+ (tumor) ma mua o nā cell EpCAM-, ʻoi aku ka kiʻekiʻe ma mua o ka hana metabolic o nā fibroblasts i loko o nā cell tumor ma TME (Kiʻi 1, E a me F).
(A a me B) Ka ikaika fluorescence waena (MFI) o ka lawe ʻana o ka glucose (2-NBDG) (A) a me ka hana mitochondrial o nā pūnaewele CD4 + T (MitoTracker ʻulaʻula ʻeleʻele) (B) Nā kiʻi hōʻike (hema) a me ka ʻikepili i hoʻopaʻa ʻia (ʻĀkau), nā pūnaewele CD8 + T a me nā pūnaewele EpCAM + CD45-tumor mai nā ascites a me ka tumor. (C) Ka lakio o nā pūnaewele CD4 + a me CD8 + (o nā pūnaewele CD3 + T) i loko o nā ascites a me ka tumor. (D) Ka nui o nā pūnaewele EpCAM + tumor i loko o nā ascites a me ka tumor (CD45−). (E a ​​me F) EpCAM + CD45-tumor a me EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) a me ka hana mitochondrial (MitoTracker ʻulaʻula ʻeleʻele) (F) nā kiʻi hōʻike (hema) a me ka ʻikepili i hoʻopaʻa ʻia (ʻĀkau) Nā ascites a me nā pūnaewele tumor. (G) Nā kiʻi hōʻike o ka hōʻike ʻana o CD25, CD137 a me PD1 e ke kahe cytometry. (H a me I) Hōʻike ʻana o CD25, CD137 a me PD1 ma nā pūnaewele CD4 + T (H) a me nā pūnaewele CD8 + T (I). (J a me K) Nā ʻano hoʻomanaʻo waena (Tcm), effector (Teff) a me ka hoʻomanaʻo effector (Tem) i hoʻokumu ʻia ma ka hōʻike ʻana o CCR7 a me CD45RO. Nā kiʻi hōʻike (hema) a me ka ʻikepili tabular (ʻākau) o nā pūnaewele CD4 + T (J) a me nā pūnaewele CD8 + T (K) i loko o nā ascites a me nā tumors. Hoʻoholo ʻia nā waiwai P e ka hoʻāʻo t-paired (*P<0.05, **P<0.01 a me ***P<0.001). Hōʻike ka laina i nā maʻi i kūlike (n = 6). FMO, fluorescence minus hoʻokahi; MFI, median fluorescence intensity.
Ua hōʻike ʻia ka loiloi hou aku i nā ʻokoʻa koʻikoʻi ʻē aʻe ma waena o ke kūlana phenotypic T cell i hoʻoholo nui ʻia. ʻOi aku ka pinepine o ka hoʻomanaʻo ʻana o ka mea i hoʻāla ʻia (Kiʻi 1, G a I) a me ka hoʻomanaʻo effector (Kiʻi 1, J a me K) i loko o nā tumors ma mua o ka ascites (ka nui o nā CD3 + T cell). Pēlā nō, ʻo ka nānā ʻana i ka phenotype ma o ka hōʻike ʻana o nā māka hoʻāla (CD25 a me CD137) a me nā māka depletion [programmed cell death protein 1 (PD1)] ua hōʻike ʻia ʻoiai he ʻokoʻa nā ʻano metabolic o kēia mau heluna kanaka (Kiʻi S1, B a E), akā ʻaʻohe ʻokoʻa metabolic koʻikoʻi i ʻike mau ʻia ma waena o nā subsets naive, effector a i ʻole memory (Kiʻi S1, F a I). ​​Ua hōʻoia ʻia kēia mau hopena ma ka hoʻohana ʻana i nā ʻano aʻo mīkini e hāʻawi aunoa i nā phenotypes cell (21), kahi i hōʻike hou aku ai i ke alo o kahi nui o nā cell bone bone (CD45 + / CD3- / CD4 + / CD45RO +) i loko o nā ascites o ka mea maʻi (Kiʻi S2A). Ma waena o nā ʻano cell āpau i ʻike ʻia, ua hōʻike kēia heluna cell myeloid i ka lawe ʻana o ka glucose kiʻekiʻe loa a me ka hana mitochondrial (Kiʻi S2, B a G). Hōʻike kēia mau hopena i nā ʻokoʻa metabolic ikaika ma waena o nā ʻano cell he nui i loaʻa i nā ascites a me nā tumors i nā maʻi HGSC.
ʻO ka pilikia nui i ka hoʻomaopopo ʻana i nā ʻano metabonomic o TIL ʻo ia ka pono e hoʻokaʻawale i nā hāpana cell T o ka maʻemaʻe, ka maikaʻi a me ka nui mai nā ʻōpū. Ua hōʻike ʻia nā haʻawina hou e hiki i nā ʻano hoʻokaʻawale a me ka hoʻonui ʻana i ka bead e pili ana i ka cytometry kahe ke alakaʻi i nā loli i nā ʻano metabolite cellular (22-24). No ka lanakila ʻana i kēia pilikia, ua hoʻomaikaʻi mākou i ke ʻano hoʻonui bead e hoʻokaʻawale a hoʻokaʻawale iā TIL mai ka maʻi ʻōpū ovarian kanaka i ʻoki ʻia ma mua o ka nānā ʻana e LC-MS/MS (e ʻike i nā Mea Hana a me nā ʻAno Hana; Kiʻi 2A). I ​​mea e loiloi ai i ka hopena holoʻokoʻa o kēia protocol ma nā loli metabolite, ua hoʻohālikelike mākou i nā ʻano metabolite o nā cell T i hoʻāla ʻia e nā mea hāʻawi olakino ma hope o ke kaʻina hoʻokaʻawale bead i luna me nā cell i hoʻokaʻawale ʻole ʻia ka bead akā noho ma ka hau. Ua ʻike kēia loiloi kaohi maikaʻi he pilina kiʻekiʻe ma waena o kēia mau kūlana ʻelua (r = 0.77), a ʻo ka hana hou ʻana o ka hui o 86 metabolites he hana hou kiʻekiʻe (Kiʻi 2B). No laila, hiki i kēia mau ʻano hana ke hana i ka loiloi metabolite pololei i loko o nā cell e hana ana i ka hoʻonui ʻana o ke ʻano cell, no laila e hāʻawi ana i ka paepae kiʻekiʻe mua no ka ʻike ʻana i nā metabolites kikoʻī ma HGSC, no laila e hiki ai i nā kānaka ke loaʻa kahi ʻike hohonu o ka polokalamu cell specificity Sexual metabolism.
(A) Kiʻikuhi schematic o ka hoʻonui ʻana i nā bead magnetic. Ma mua o ka nānā ʻana e LC-MS/MS, e hele nā ​​​​​​pūnaewele i ʻekolu mau pōʻai o ka hoʻonui ʻana i nā bead magnetic a i ʻole e noho ma luna o ka hau. (B) Ka hopena o ke ʻano hoʻonui ma ka nui o nā metabolites. ʻO ka awelika o ʻekolu mau ana no kēlā me kēia ʻano hoʻonui ± SE. Hōʻike ka laina hina i kahi pilina 1: 1. ʻO ka pilina intra-class (ICC) o nā ana i hana hou ʻia i hōʻike ʻia ma ka lepili axis. NAD, nicotinamide adenine dinucleotide. (C) Kiʻikuhi schematic o ke kahe hana o ka nānā ʻana i ka metabolite maʻi. Hōʻiliʻili ʻia nā ascites a i ʻole nā ​​​​​​tumors mai nā maʻi a cryopreserved. Ua kālailai ʻia kahi hapa liʻiliʻi o kēlā me kēia laʻana e ka cytometry kahe, ʻoiai ua hele nā ​​​​​​laʻana i koe i ʻekolu mau pōʻai o ka hoʻonui ʻana no nā cell CD4+, CD8+ a me CD45-. Ua kālailai ʻia kēia mau ʻāpana cell me ka hoʻohana ʻana iā LC-MS/MS. (D) Palapala wela o ka nui o ka metabolite maʻamau. Hōʻike ka dendrogram i ka hui ʻana o Ward o nā mamao Euclidean ma waena o nā laʻana. (E) Ka loiloi ʻāpana nui (PCA) o ka palapala ʻāina metabolite laʻana, e hōʻike ana i ʻekolu mau replicates o kēlā me kēia laʻana, ua hoʻopili ʻia nā laʻana mai ka mea maʻi like e kahi laina. (F) ʻO ka PCA o ka ʻikepili metabolite o ka laʻana i hoʻoponopono ʻia ma ka mea maʻi (ʻo ia hoʻi, me ka hoʻohana ʻana i ka redundancy hapa); ua kaupalena ʻia ke ʻano laʻana e ka hull convex. PC1, ʻāpana nui 1; PC2, ʻāpana nui 2.
ʻO ka mea aʻe, ua hoʻohana mākou i kēia ʻano hoʻonui e kālailai i 99 metabolites i loko o nā ʻāpana CD4 +, CD8 + a me CD45-cell i loko o nā ascites mua a me nā tumors o ʻeono mau maʻi HGSC (Kiʻi 2C, Kiʻi S3A a me ka Papa S3 a me S4). ʻO ka heluna kanaka hoihoi no 2% a 70% o ka laʻana nui mua o nā cell ola, a ʻokoʻa loa ka hapa o nā cell ma waena o nā maʻi. Ma hope o ka hoʻokaʻawale ʻana i nā beads, ʻo ka hapa hoʻonui o ka hoihoi (CD4 +, CD8 + a i ʻole CD45-) e helu ana ma mua o 85% o nā cell ola āpau i loko o ka laʻana ma ka awelika. ʻAe kēia ʻano hoʻonui iā mākou e kālailai i nā heluna cell mai ka metabolism o nā ʻiʻo tumor kanaka, kahi mea hiki ʻole ke hana mai nā laʻana nui. Ma ka hoʻohana ʻana i kēia protocol, ua hoʻoholo mākou ua hoʻokiʻekiʻe ʻia kēia mau metabolites immunosuppressive ʻelua i ʻike maikaʻi ʻia i loko o nā cell T tumor a i ʻole nā ​​​​cell tumor (Kiʻi S3, B a me C). No laila, hōʻike kēia mau hopena i ka ʻoiaʻiʻo a me ka hiki o kā mākou hoʻokaʻawale cell a me ka ʻenehana spectrometry mass e ʻike i nā metabolites koʻikoʻi biologically i loko o nā ʻiʻo maʻi.
Ua hōʻike pū kā mākou loiloi i kahi kaʻawale metabolic ikaika o nā ʻano cell i loko a ma waena o nā mea maʻi (Kiʻi 2D a me Kiʻi S4A). Ma ke ʻano kūikawā, i hoʻohālikelike ʻia me nā mea maʻi ʻē aʻe, ua hōʻike ka mea maʻi 70 i nā ʻano metabolic like ʻole (Kiʻi 2E a me Kiʻi S4B), e hōʻike ana he nui paha ka heterogeneity metabolic ma waena o nā mea maʻi. He mea kūpono ke hoʻomaopopo ʻana i ka hoʻohālikelike ʻia me nā mea maʻi ʻē aʻe (1.2 a 2 lita; Papa S1), ʻoi aku ka liʻiliʻi o ka nui o nā ascites i hōʻiliʻili ʻia ma ka mea maʻi 70 (80 ml). ʻO ka kaohi ʻana o ka heterogeneity ma waena o nā mea maʻi i ka wā o ka loiloi ʻāpana nui (no ka laʻana, me ka hoʻohana ʻana i ka loiloi redundancy hapa) e hōʻike ana i nā loli mau ma waena o nā ʻano cell, a ua hōʻuluʻulu maopopo ʻia nā ʻano cell a/a i ʻole microenvironment e like me ka metabolite profile (Kiʻi 2F). Ua hoʻokūpaʻa ka loiloi o nā metabolites hoʻokahi i kēia mau hopena a ua hōʻike i nā ʻokoʻa koʻikoʻi ma waena o nā ʻano cell a me ka microenvironment. He mea kūpono ke hoʻomaopopo ʻana ʻo ka ʻokoʻa koʻikoʻi loa i ʻike ʻia ʻo MNA, ka mea i hoʻonui pinepine ʻia i nā cell CD45- a me nā cell CD4+ a me CD8+ e komo i loko o ka puʻupuʻu (Kiʻi 3A). No nā pūnaewele CD4 +, ʻike nui ʻia kēia hopena, a ʻike ʻia hoʻi ua hoʻopilikia nui ʻia ka MNA i loko o nā pūnaewele CD8 + e ke kaiapuni. Eia naʻe, ʻaʻole koʻikoʻi kēia, no ka mea, ʻekolu wale nō o nā maʻi ʻeono i hiki ke loiloi ʻia no nā helu CD8+ tumor. Ma waho aʻe o MNA, i loko o nā ʻano pūnaewele like ʻole i loko o nā ascites a me nā tumors, ʻo nā metabolites ʻē aʻe i hōʻike maikaʻi ʻole ʻia ma TIL he waiwai like ʻole hoʻi (Nā Kiʻi S3 a me S4). No laila, hōʻike kēia ʻikepili i kahi hoʻonohonoho hoʻohiki o nā metabolites immunomodulatory no ka noiʻi hou aku.
(A) Ka ʻike maʻamau o MNA i loko o nā pūnaewele CD4+, CD8+ a me CD45- mai nā ascites a me ka tumor. Hōʻike ka pahu pahu i ka median (laina), ka laulā interquartile (frame hinge) a me ka laulā ʻikepili, a hiki i ka 1.5 mau manawa o ka laulā interquartile (frame whisker). E like me ka mea i wehewehe ʻia ma nā Mea Hana a me nā Hana o ka Maʻi, e hoʻohana i ka waiwai limma o ka mea maʻi e hoʻoholo ai i ka waiwai P (*P<0.05 a me **P<0.01). (B) Kiʻikuhi schematic o ka metabolism MNA (60). Metabolites: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl-2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide ribose; NMN, nicotinamide mononucleotide. Nā ʻenzymes (ʻōmaʻomaʻo): NNMT, nicotinamide N-methyltransferase; SIRT, sirtuins; NAMPT, nicotinamide phosphoribosyl transferase; AOX1, aldehyde oxidase 1; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mono Nucleotide adenylate transferase; Pnp1, purine nucleoside phosphorylase. (C) t-SNE o scRNA-seq o ascites (hina) a me ka tumor (ʻulaʻula; n = 3 mau maʻi). (D) Hōʻike ʻana o NNMT i nā heluna cell like ʻole i ʻike ʻia me ka hoʻohana ʻana i ka scRNA-seq. (E) Hōʻike ʻana o NNMT a me AOX1 ma SK-OV-3, ka puʻupaʻa embryonic kanaka (HEK) 293T, nā pūnaewele T a me nā pūnaewele T i mālama ʻia me MNA. Hōʻike ʻia ka hōʻike i pelu ʻia e pili ana iā SK-OV-3. Hōʻike ʻia ke ʻano hōʻike me SEM (n = 6 mau mea hāʻawi olakino). ʻO nā waiwai Ct ʻoi aku ma mua o 35 i manaʻo ʻia ʻaʻole hiki ke ʻike ʻia (UD). (F) Hōʻike ʻana o SLC22A1 a me SLC22A2 ma SK-OV-3, HEK293T, nā pūnaewele T a me nā pūnaewele T i mālama ʻia me 8mM MNA. Hōʻike ʻia ka hōʻike i pelu ʻia e pili ana iā SK-OV-3. Hōʻike ʻia ke ʻano hōʻike me SEM (n = 6 mau mea hāʻawi olakino). ʻO nā waiwai Ct ʻoi aku ma mua o 35 i manaʻo ʻia ʻaʻole hiki ke ʻike ʻia (UD). (G) ʻO ka nui o ka MNA o ke kelepona i loko o nā pūnaewele T hāʻawi olakino i hoʻāla ʻia ma hope o 72 mau hola o ka hoʻoulu ʻana me MNA. Hōʻike ʻia ke ʻano hōʻike me SEM (n = 4 mau mea hāʻawi olakino).
Hoʻopuka ʻia ʻo MNA ma ka hoʻoili ʻana i ka hui methyl mai S-adenosyl-1-methionine (SAM) i nicotinamide (NA) e ka nicotinamide N-methyltransferase (NNMT; Kiʻi 3B). Hoʻonui nui ʻia ʻo NNMT i nā ʻano maʻi kanesa kanaka a pili pū me ka hoʻonui ʻana, ka hoʻouka kaua ʻana a me ka metastasis (25-27). No ka hoʻomaopopo maikaʻi ʻana i ke kumu o MNA i loko o nā pūnaewele T ma TME, ua hoʻohana mākou i ka scRNA-seq e wehewehe i ka hōʻike ʻana o NNMT ma waena o nā ʻano pūnaewele i loko o nā ascites a me nā tumors o ʻekolu mau maʻi HGSC (Papa S5). Ua hōʻike ʻia ka nānā ʻana o kahi 6,500 mau pūnaewele i loko o nā ascites a me nā wahi tumor, ua kaupalena ʻia ka hōʻike ʻana o NNMT i nā heluna fibroblast a me nā cell tumor i manaʻo ʻia (Kiʻi 3, C a me D). He mea kūpono ke hoʻomaopopo ʻana ʻaʻohe hōʻike NNMT maopopo i loko o kekahi heluna kanaka e hōʻike ana i ka PTPRC (CD45 +) (Kiʻi 3D a me Kiʻi S5A), e hōʻike ana ua hoʻokomo ʻia ka MNA i ʻike ʻia i loko o ka metabolite spectrum i loko o nā pūnaewele T. ʻO ka hōʻike ʻana o ka aldehyde oxidase 1 (AOX1) e hoʻololi i ka MNA i 1-methyl-2-pyridone-5-carboxamide (2-PYR) a i ʻole 1-methyl-4-pyridone-5-carboxamide (4-PYR); Kiʻi 3B) ua kaupalena ʻia hoʻi i ka heluna o nā fibroblasts e hōʻike ana iā COL1A1 (Kiʻi S5A), kahi e hōʻike pū ana ʻaʻole i loaʻa i nā pūnaewele T ka hiki o ka metabolism MNA maʻamau. Ua hōʻoia ʻia ke ʻano hōʻike o kēia mau genes e pili ana iā MNA me ka hoʻohana ʻana i kahi ʻikepili pūnaewele kūʻokoʻa ʻelua mai nā ascites mai nā maʻi HGSC (Kiʻi S5B; n = 6) (16). Eia kekahi, ua hōʻike ka loiloi quantitative polymerase chain reaction (qPCR) o nā pūnaewele T hāʻawi olakino i mālama ʻia me MNA i ka hoʻohālikelike ʻana me nā pūnaewele tumor ovarian SK-OV-3, ʻaneʻane ʻaʻole i hōʻike ʻia ʻo NNMT a i ʻole AOX1 (Kiʻi 3E). Hōʻike kēia mau hopena i manaʻo ʻole ʻia e hiki ke hūnā ʻia ʻo MNA mai nā fibroblasts a i ʻole nā ​​tumors i loko o nā pūnaewele T pili ma TME.
ʻOiai ʻo nā moho e komo pū ana me ka ʻohana o nā mea lawe cation organik 1 a 3 (OCT1, OCT2 a me OCT3) i hoʻopili ʻia e ka ʻohana mea lawe soluble 22 (SLC22) (SLC22A1, SLC22A2 a me SLC22A3), ʻaʻole i wehewehe ʻia nā mea lawe hiki o MNA (28). Ua hōʻike ʻo QPCR o mRNA mai nā cell T donor olakino i nā pae haʻahaʻa o ka hōʻike ʻana o SLC22A1 akā ʻaʻole hiki ke ʻike ʻia o SLC22A2, kahi i hōʻoia ai ua hōʻike mua ʻia ma ka palapala (Kiʻi 3F) (29). I ka hoʻohālikelike ʻana, ua hōʻike ka laina cell tumor ovarian SK-OV-3 i nā pae kiʻekiʻe o nā mea lawe ʻelua (Kiʻi 3F).
I mea e hoʻāʻo ai i ka hiki ke loaʻa i nā pūnaewele T ke komo i ka MNA haole, ua hoʻoulu ʻia nā pūnaewele T hāʻawi olakino no 72 mau hola i ke alo o nā ʻano like ʻole o ka MNA. I ka loaʻa ʻole o ka MNA exogenous, ʻaʻole hiki ke ʻike ʻia ka ʻike cellular o MNA (Kiʻi 3G). Eia nō naʻe, ua hōʻike nā pūnaewele T i hoʻāla ʻia i mālama ʻia me ka MNA exogenous i ka piʻi ʻana o ka nui o ka MNA i loko o nā pūnaewele, a hiki i ka 6 mM MNA (Kiʻi 3G). Hōʻike kēia hopena ʻoiai ke haʻahaʻa o ka hōʻike ʻana o ka transporter a me ka nele o ka enzyme nui nāna e kuleana no ka metabolism MNA intracellular, hiki iā TIL ke lawe i ka MNA.
ʻO ka spectrum o nā metabolites i loko o nā cell T o nā maʻi a me nā hoʻokolohua hoʻomoʻa MNA in vitro e hoʻonui i ka hiki ke hoʻokuʻu ʻia e nā fibroblasts pili i ka maʻi kanesa (CAF) ka MNA a hiki i nā cell tumor ke hoʻoponopono i ke ʻano a me ka hana o TIL. No ka hoʻoholo ʻana i ka hopena o MNA ma nā cell T, ua hoʻāla ʻia nā cell T donor olakino in vitro i ke alo a i ʻole ka ʻole o MNA, a ua loiloi ʻia ko lākou ulu ʻana a me ka hana cytokine. Ma hope o 7 mau lā o ka hoʻohui ʻana i ka MNA ma ka nui kiʻekiʻe loa, ua hoʻemi iki ʻia ka helu pālua o ka heluna kanaka, ʻoiai ua mālama ʻia ka ikaika ma nā doses āpau (Kiʻi 4A). Eia kekahi, ʻo ka mālama ʻana i ka MNA exogenous i hopena i ka hoʻonui ʻana i ka hapa o nā cell CD4 + a me CD8 + T e hōʻike ana i ka tumor necrosis factor-α (TNFα; Kiʻi 4B). I ka hoʻohālikelike ʻana, ua hoʻemi nui ʻia ka hana intracellular o IFN-γ i nā cell CD4 + T, akā ʻaʻole i nā cell CD8 + T, a ʻaʻohe loli koʻikoʻi i ka interleukin 2 (IL-2; Kiʻi 4, C a me D). No laila, ua hōʻike ka enzyme-linked immunosorbent assay (ELISA) o nā supernatants mai kēia mau moʻomeheu T cell i mālama ʻia e MNA i ka piʻi nui ʻana o TNFα, ka emi ʻana o IFN-γ, a ʻaʻohe loli o IL-2 (Kiʻi 4, E a i G). . ʻO ka emi ʻana o IFN-γ e hōʻike ana he kuleana ko MNA i ka pale ʻana i ka hana anti-tumor o nā cell T. I mea e hoʻohālike ai i ka hopena o MNA ma ka cytotoxicity i hoʻopili ʻia e nā cell T, nā cell chimeric antigen receptor T (FRα-CAR-T) e kuhikuhi ana i ka folate receptor α a me CAR-T (GFP) i hoʻoponopono ʻia e nā cell fluorescent green (GFP) -CAR-T) i hana ʻia e nā cell mononuclear koko peripheral donor olakino (PBMC). Ua ulu ʻia nā cell CAR-T no 24 mau hola i ke alo o MNA, a laila ua ulu pū ʻia me nā cell tumor ovarian SK-OV-3 kanaka e hōʻike ana i ka folate receptor α ma kahi effector i ka target ratio o 10:1. Ua hopena ka mālama ʻana o MNA i ka emi nui ʻana o ka hana pepehi kanaka o nā pūnaewele FRα-CAR-T, kahi i like me nā pūnaewele FRα-CAR-T i mālama ʻia me ka adenosine (Kiʻi 4H).
(A) Ka huina o nā cell ola a me ka pāpālua ʻana o ka heluna kanaka (PD) pololei mai ka moʻomeheu i ka lā 7. Hōʻike ka pakuhi pā i ka awelika + SEM o ʻeono mau mea hāʻawi olakino. Hōʻike i ka ʻikepili mai ka liʻiliʻi he n = 3 mau hoʻokolohua kūʻokoʻa. (B a i D) Ua hoʻohana ʻia ʻo CD3/CD28 a me IL-2 e hoʻoulu i nā cell T ma kā lākou mau MNA no 7 mau lā. Ma mua o ka nānā ʻana, ua hoʻoulu ʻia nā cell me PMA/ionomycin me GolgiStop no 4 mau hola. Hōʻike ʻana o TNFα (B) i nā cell T. Kiʻi hoʻohālike (hema) a me ka ʻikepili tabular (ʻākau) o ka hōʻike ʻana o TNFα i nā cell ola. Hōʻike ʻana o IFN-γ (C) a me IL-2 (D) i nā cell T. Ua ana ʻia ka hōʻike ʻana o nā cytokines e ka cytometry kahe. Hōʻike ka pakuhi pā i ka awelika (n = 6 mau mea hāʻawi olakino) + SEM. E hoʻohana i ka loiloi hoʻokahi ala o ka variance a me nā ana i hana hou ʻia (*P<0.05 a me **P<0.01) e hoʻoholo i ka waiwai P. Hōʻike i ka ʻikepili mai ka liʻiliʻi he n = 3 mau hoʻokolohua kūʻokoʻa. (E a i G) Ua hoʻohana ʻia ʻo CD3/CD28 a me IL-2 e hoʻoulu i nā pūnaewele T ma kā lākou mau kikowaena MNA no 7 mau lā. Ua hōʻiliʻili ʻia ka medium ma mua a ma hope o 4 mau hola o ka hoʻoulu ʻana o PMA/ionomycin. Ua ana ʻia nā kikowaena o TNFα (E), IFN-γ (F) a me IL-2 (G) e ELISA. Hōʻike ka pakuhi pā i ka awelika (n = 5 mau mea hāʻawi olakino) + SEM. Ua hoʻoholo ʻia ka waiwai P me ka hoʻohana ʻana i ka loiloi hoʻokahi ala o ka variance a me nā ana hou (*P<0.05). Hōʻike ka laina kiko i ka palena ʻike o ka ʻike. (H) Hoʻāʻo lysis cell. Ua hoʻoponopono ʻia nā pūnaewele FRα-CAR-T a i ʻole GFP-CAR-T me adenosine (250μM) a i ʻole MNA (10 mM) no 24 mau hola, a i ʻole waiho ʻia me ka mālama ʻole ʻia (Ctrl). Ua ana ʻia ka pakeneka pepehi ʻana o nā pūnaewele SK-OV-3. Ua hoʻoholo ʻia ka waiwai P e ka hoʻāʻo Welch t (*P<0.5 a me **P<0.01).
I mea e loaʻa ai kahi ʻike mechanistic o ka hoʻoponopono ʻana o ka hōʻike ʻana o TNFα e hilinaʻi ana iā MNA, ua loiloi ʻia nā loli i ka TNFα mRNA o nā cell T i mālama ʻia me MNA (Kiʻi 5A). Ua hōʻike nā cell T hāʻawi olakino i mālama ʻia me MNA i ka piʻi ʻelua manawa i nā pae transcription TNFα, e hōʻike ana ua hilinaʻi ʻo MNA i ka hoʻoponopono transcriptional TNFα. No ka noiʻi ʻana i kēia ʻano hoʻoponopono hiki, ua loiloi ʻia ʻelua mau mea transcription i ʻike ʻia e hoʻoponopono iā TNFα, ʻo ia hoʻi ka activated T cell nuclear factor (NFAT) a me ka protein kikoʻī 1 (Sp1), ma ke ʻano he pane i ka hoʻopaʻa ʻana o MNA i ka mea hoʻolaha TNFα proximal (30). Loaʻa i ka mea hoʻolaha TNFα he 6 mau wahi hoʻopaʻa NFAT i ʻike ʻia a me 2 mau wahi hoʻopaʻa Sp1, e uhi ana ma kahi hoʻokahi [-55 mau hui kumu (bp) mai ka 5'cap] (30). Ua hōʻike ʻo Chromatin immunoprecipitation (ChIP) i ka wā i mālama ʻia ai me MNA, ua hoʻonui ʻekolu manawa ka hoʻopaʻa ʻana o Sp1 i ka mea hoʻolaha TNFα. Ua hoʻonui pū ʻia ka hoʻohui ʻana o NFAT a ua kokoke i ke koʻikoʻi (Kiʻi 5B). Hōʻike kēia ʻikepili e hoʻoponopono ana ʻo MNA i ka hōʻike ʻana o TNFα ma o ka palapala Sp1, a i kahi liʻiliʻi ka hōʻike ʻana o NFAT.
(A) Ke hoʻohālikelike ʻia me nā pūnaewele T i hoʻoulu ʻia me ka ʻole o MNA, ka loli o ka hoʻopuka ʻana o TNFα i nā pūnaewele T i mālama ʻia me MNA. Hōʻike ʻia ke ʻano hoʻopuka me SEM (n = 5 mau mea hāʻawi olakino). Hōʻike i ka ʻikepili mai ka liʻiliʻi he n = 3 mau hoʻokolohua kūʻokoʻa. (B) ʻO ka mea hoʻolaha TNFα o nā pūnaewele T i mālama ʻia me a i ʻole me ka ʻole o 8 mM MNA ma hope o NFAT a me Sp1 i hui pū ʻia me (Ctrl) a me ka hoʻoulu ʻana o PMA/ionomycin no 4 mau hola. Ua hoʻohana ʻia ʻo Immunoglobulin G (IgG) a me H3 ma ke ʻano he mau kaohi maikaʻi ʻole a maikaʻi no ka immunoprecipitation, kēlā me kēia. Ua hōʻike ka helu ʻana o ChIP ua hoʻonui ʻia ka nakinaki ʻana o Sp1 a me NFAT i ka mea hoʻolaha TNFα i nā pūnaewele i mālama ʻia me MNA i nā manawa he nui i hoʻohālikelike ʻia me ka kaohi. Hōʻike i ka ʻikepili mai ka liʻiliʻi he n = 3 mau hoʻokolohua kūʻokoʻa. ʻO ka waiwai P i hoʻoholo ʻia e nā hoʻāʻo t he nui (*** P <0.01). (C) Ke hoʻohālikelike ʻia me nā ascites o HGSC, ua hōʻike nā pūnaewele T (ʻaʻole cytotoxic) i ka hoʻonui ʻia o ka hōʻike ʻana o TNF i loko o ka puʻupuʻu. Hōʻike nā kala i nā maʻi like ʻole. Ua hoʻokaʻawale ʻia nā pūnaewele i hōʻike ʻia i 300 a ua hoʻoluliluli ʻia e kaupalena i ka overdrawing (** Padj = 0.0076). (D) Kumu hoʻohālike i manaʻo ʻia no ka maʻi ʻaʻai ovarian. Hana ʻia ka MNA i loko o nā pūnaewele tumor a me nā fibroblasts ma TME a lawe ʻia e nā pūnaewele T. Hoʻonui ka MNA i ka nakinaki ʻana o Sp1 i ka mea hoʻolaha TNFα, e alakaʻi ana i ka hoʻonui ʻana i ka transcription TNFα a me ka hana cytokine TNFα. Hoʻoulu pū ka MNA i ka emi ʻana o IFN-γ. ʻO ka pale ʻana i ka hana o nā pūnaewele T e alakaʻi i ka emi ʻana o ka hiki ke pepehi a me ka wikiwiki o ka ulu ʻana o ka tumor.
Wahi a nā hōʻike, loaʻa iā TNFα nā hopena anti-tumor a me nā hopena anti-tumor e hilinaʻi mua a hope, akā he kuleana koʻikoʻi kona i ka hoʻolaha ʻana i ka ulu ʻana a me ka metastasis o ka maʻi ʻaʻai ovarian (31-33). Wahi a nā hōʻike, ʻoi aku ka nui o ka TNFα i loko o nā ascites a me nā ʻiʻo tumor i nā maʻi me ka maʻi ʻaʻai ovarian ma mua o nā ʻiʻo benign (34-36). Ma ke ʻano o ka mīkini, hiki iā TNFα ke hoʻoponopono i ka hoʻoulu ʻana, ka hana a me ka hoʻonui ʻana o nā keʻena koko keʻokeʻo, a hoʻololi i ke ʻano o nā keʻena kanesa (37, 38). E kūlike me kēia mau ʻike, ua hōʻike ka loiloi hōʻike gene ʻokoʻa ua hoʻonui nui ʻia ʻo TNF i loko o nā keʻena T i loko o nā ʻiʻo tumor i hoʻohālikelike ʻia me nā ascites (Kiʻi 5C). ʻIke wale ʻia ka hoʻonui ʻana o ka hōʻike ʻana o TNF i nā heluna cell T me kahi phenotype non-cytotoxic (Kiʻi S5A). I ​​ka hōʻuluʻulu manaʻo, kākoʻo kēia mau ʻikepili i ka manaʻo he mau hopena immunosuppressive a me ka hoʻolaha ʻana o ka tumor ko MNA ma HGSC.
ʻO ka lepili fluorescent e pili ana i ka cytometry kahe ua lilo i ke ʻano nui no ke aʻo ʻana i ka metabolism TIL. Ua hōʻike kēia mau haʻawina i ka hoʻohālikelike ʻia me nā lymphocytes koko peripheral a i ʻole nā ​​​​​​pūnaewele T mai nā ʻōpū lymphoid lua, ʻoi aku ka kiʻekiʻe o ka TIL murine a me ke kanaka e lawe i ka glucose (4, 39) a me ka nalowale mālie o ka hana mitochondrial (19, 40). ʻOiai ua ʻike mākou i nā hopena like ma kēia haʻawina, ʻo ke kumu nui o ka hoʻomohala ʻana, ʻo ia ka hoʻohālikelike ʻana i ka metabolism o nā pūnaewele tumor a me TIL mai ka ʻiʻo tumor like i ʻoki ʻia. E like me kekahi o kēia mau hōʻike mua, ʻoi aku ka kiʻekiʻe o ka lawe ʻana o ka glucose i nā pūnaewele tumor (CD45-EpCAM +) mai nā ascites a me nā tumors ma mua o nā pūnaewele CD8 + a me CD4 + T, e kākoʻo ana i ka hiki ke hoʻohālikelike ʻia ka lawe ʻana o ka glucose kiʻekiʻe o nā pūnaewele tumor me nā pūnaewele T. ʻO ke kumumanaʻo o ka hoʻokūkū pūnaewele T. TME. Eia naʻe, ʻoi aku ka kiʻekiʻe o ka hana mitochondrial o nā pūnaewele tumor ma mua o nā pūnaewele CD8 + T, akā ua like ka hana mitochondrial me nā pūnaewele CD4 + T. Hoʻoikaika kēia mau hopena i ke kumumanaʻo e kū mai ana he mea nui ka metabolism oxidative no nā pūnaewele tumor (41, 42). Manaʻo pū lākou he ʻoi aku ka maʻalahi o nā pūnaewele CD8 + T i ka hana oxidative ma mua o nā pūnaewele CD4 + T, a i ʻole e hoʻohana paha nā pūnaewele CD4 + T i nā kumu kalapona ʻē aʻe ma mua o ka glucose e mālama i ka hana mitochondrial (43, 44). Pono e hoʻomaopopo ʻia ʻaʻole mākou i ʻike i kahi ʻokoʻa i ka lawe ʻana o ka glucose a i ʻole ka hana mitochondrial ma waena o nā mea hoʻokō CD4 + T, ka hoʻomanaʻo T effector a me nā pūnaewele hoʻomanaʻo T waena i nā ascites. Pēlā nō, ʻaʻohe pili o ke kūlana hoʻokaʻawale o nā pūnaewele CD8 + T i loko o nā tumors me nā loli i ka lawe ʻana o ka glucose, e hōʻike ana i ka ʻokoʻa koʻikoʻi ma waena o nā pūnaewele T i mahi ʻia i loko o vitro a me ke kanaka TIL in vivo (22). Ua hōʻoia ʻia kēia mau nānā ʻana e ka hoʻohana ʻana i ka hoʻokaʻawale ʻana o ka heluna kanaka cell automatic unbiased, kahi i hōʻike hou aku ai he nui nā pūnaewele CD45 + / CD3- / CD4 + / CD45RO + me ka lawe ʻana o ka glucose kiʻekiʻe a me ka hana mitochondrial ma mua o nā pūnaewele tumor akā he heluna cell Metabolic active. Hiki i kēia heluna ke hōʻike i ka subpopulation putative o nā pūnaewele suppressor myeloid a i ʻole nā ​​​​pūnaewele dendritic plasmacytoid i ʻike ʻia ma ka loiloi scRNA-seq. ʻOiai ua hōʻike ʻia kēia mau mea ʻelua i loko o nā ʻōpū ovarian kanaka [45], pono nō lākou i ka hana hou aʻe e wehewehe i kēia myeloid subpopulation.
ʻOiai hiki i nā ʻano hana e pili ana i ka cytometry kahe ke wehewehe i nā ʻokoʻa maʻamau i ka glucose a me ka metabolism oxidative ma waena o nā ʻano cell, ʻaʻole i hoʻoholo ʻia nā metabolites pololei i hana ʻia e ka glucose a i ʻole nā ​​​​kumu kalapona ʻē aʻe no ka metabolism mitochondrial ma TME. ʻO ka hāʻawi ʻana i ke alo a i ʻole ka ʻole o nā metabolites i kahi subset TIL i hāʻawi ʻia e pono ai ka hoʻomaʻemaʻe ʻana o ka heluna cell mai ka ʻiʻo i ʻoki ʻia. No laila, ʻo kā mākou ʻano hoʻonui cell i hui pū ʻia me ka spectrometry mass hiki ke hāʻawi i nā ʻike i nā metabolites i hoʻonui ʻokoʻa ʻia i loko o nā cell T a me nā heluna cell tumor i nā laʻana maʻi e hoʻohālikelike ana. ʻOiai he mau pono kēia ʻano hana ma mua o ka fluorescence-activated cell sorting, hiki ke hoʻopilikia ʻia kekahi mau hale waihona puke metabolite ma muli o ke kūpaʻa kūlohelohe a/a i ʻole ka wikiwiki o ka huli ʻana (22). Eia naʻe, ua hiki i kā mākou ʻano hana ke ʻike i ʻelua mau metabolites immunosuppressive i ʻike ʻia, adenosine a me kynurenine, no ka mea, ʻokoʻa loa lākou ma waena o nā ʻano laʻana.
Hāʻawi kā mākou loiloi metabonomic o nā tumors a me nā subtypes TIL i nā ʻike hou aku i ke kuleana o nā metabolites i ka ovarian TME. ʻO ka mea mua, me ka hoʻohana ʻana i ka cytometry kahe, ua hoʻoholo mākou ʻaʻohe ʻokoʻa o ka hana mitochondrial ma waena o nā tumors a me nā cell CD4 + T. Eia nō naʻe, ua hōʻike ka loiloi LC-MS/MS i nā loli koʻikoʻi i ka nui o nā metabolites i waena o kēia mau heluna kanaka, e hōʻike ana he pono ka wehewehe akahele i nā hopena e pili ana i ka metabolism TIL a me kāna hana metabolic holoʻokoʻa. ʻO ka lua, ʻo MNA ka metabolite me ka ʻokoʻa nui loa ma waena o nā cell CD45 a me nā cell T i nā ascites, ʻaʻole nā ​​​​tumors. No laila, ʻo ka compartmentalization a me ka wahi o ka tumor he mau hopena like ʻole i ka metabolism TIL, kahi e hōʻike ana i ka heterogeneity hiki i kahi microenvironment i hāʻawi ʻia. ʻO ke kolu, ʻo ka hōʻike ʻana o ka enzyme hana MNA NNMT ua kaupalena nui ʻia i ka CAF, ʻo ia nā cell tumor i kahi liʻiliʻi, akā ʻike ʻia nā pae MNA hiki ke ʻike ʻia i nā cell T i loaʻa mai ka tumor. ʻO ka overexpression o NNMT i ka ovarian CAF he hopena hoʻolaha kanesa i ʻike ʻia, ma muli o ka hoʻolaha ʻana o ka metabolism CAF, ka hoʻouka kaua tumor a me ka metastasis (27). ʻOiai ke kaulike o ka pae holoʻokoʻa o TIL, pili loa ka hōʻike ʻana o NNMT ma CAF i ka subtype mesenchymal Cancer Genome Atlas (TCGA), kahi e pili ana me ka prognosis maikaʻi ʻole (27, 46, 47). ʻO ka mea hope loa, ʻo ka hōʻike ʻana o ka enzyme AOX1 kuleana no ka hoʻohaʻahaʻa ʻana o MNA ua kaupalena ʻia hoʻi i ka heluna kanaka CAF, e hōʻike ana ʻaʻole hiki i nā pūnaewele T ke hoʻololi i ka MNA. Kākoʻo kēia mau hopena i ka manaʻo ʻoiai e pono ana ka hana hou e hōʻoia i kēia ʻike, hiki i nā pae kiʻekiʻe o MNA i nā pūnaewele T ke hōʻike i ke alo o kahi microenvironment CAF immunosuppressive.
Ma muli o ka pae haʻahaʻa o ka hōʻike ʻana o nā mea lawe MNA a me nā pae ʻike ʻole ʻia o nā protein koʻikoʻi e pili ana i ka metabolism MNA, ʻo ka loaʻa ʻana o MNA i loko o nā pūnaewele T he mea i manaʻo ʻole ʻia. ʻAʻole hiki ke ʻike ʻia ʻo NNMT a me AOX1 e ka nānā ʻana o scRNA-seq a me ka qPCR i kuhikuhi ʻia o ʻelua mau cohorts kūʻokoʻa. Hōʻike kēia mau hopena ʻaʻole i hana ʻia ʻo MNA e nā pūnaewele T, akā ua omo ʻia mai ka TME a puni. Hōʻike nā hoʻokolohua in vitro e hōʻiliʻili pinepine ana nā pūnaewele T i ka MNA exogenous.
Ua hōʻike ʻia kā mākou mau haʻawina in vitro e hoʻoulu ka MNA exogenous i ka hōʻike ʻana o TNFα i loko o nā pūnaewele T a hoʻonui i ka hoʻopaʻa ʻana o Sp1 i ka mea hoʻolaha TNFα. ʻOiai he mau hana anti-tumor a me anti-tumor ko TNFα, i loko o ke kanesa ovarian, hiki iā TNFα ke hoʻoulu i ka ulu ʻana o ke kanesa ovarian (31-33). ʻO ka neutralization o TNFα i loko o ka moʻomeheu cell tumor ovarian a i ʻole ka hoʻopau ʻana i ka hōʻailona TNFα i loko o nā hiʻohiʻona ʻiole hiki ke hoʻomaikaʻi i ka hana cytokine inflammatory i hoʻopili ʻia e TNFα a kāohi i ka ulu ʻana o ka tumor (32, 35). No laila, i kēia hihia, hiki i ka MNA i loaʻa mai TME ke hana ma ke ʻano he metabolite pro-inflammatory ma o kahi ʻano hana TNFα-dependent ma o ka autocrine loop, no laila e hoʻolaha ana i ka hanana a me ka laha ʻana o ke kanesa ovarian (31). Ma muli o kēia hiki, ke aʻo ʻia nei ka TNFα blockade ma ke ʻano he mea lapaʻau kūpono no ke kanesa ovarian (37, 48, 49). Eia kekahi, hoʻopilikia ka MNA i ka cytotoxicity o nā cell CAR-T i nā cell tumor ovarian, e hāʻawi ana i nā hōʻike hou aku no ka hoʻopau ʻana o ka pale ʻana o MNA. Ma ke ʻano hui pū ʻia, hōʻike kēia mau hopena i kahi kumu hoʻohālike kahi e hoʻokuʻu ai nā ʻōpū a me nā cell CAF i ka MNA i loko o ka TME extracellular. Ma o (i) ka hoʻoulu ʻana o ka ulu ʻana o ka maʻi ʻōpū ovarian i hoʻokomo ʻia e TNF a me (ii) ka pale ʻana i ka hana cytotoxic cell T i hoʻokomo ʻia e MNA, loaʻa paha i kēia kahi hopena ʻelua o ka ʻōpū (Kiʻi 5D).
I ka hopena, ma ka hoʻopili ʻana i ka hui pū ʻana o ka hoʻonui wikiwiki ʻana o nā cell, ka hoʻonohonoho ʻana o nā cell hoʻokahi a me ka metabolic profiling, ua hōʻike kēia haʻawina i nā ʻokoʻa immunometabolomic nui ma waena o nā tumors a me nā ascites cell i nā maʻi HGSC. Ua hōʻike kēia loiloi piha he mau ʻokoʻa i ka lawe ʻana o ka glucose a me ka hana mitochondrial ma waena o nā cell T, a ua ʻike ʻia ʻo MNA ma ke ʻano he metabolite hoʻoponopono pale ʻole-cell autonomous. Loaʻa i kēia ʻikepili ka hopena i ke ʻano o ka hopena o TME i ka metabolism cell T i nā maʻi kanesa kanaka. ʻOiai ua hōʻike ʻia ka hoʻokūkū pololei no nā meaʻai ma waena o nā cell T a me nā cell cancer, hiki i nā metabolites ke hana ma ke ʻano he mau mea hoʻoponopono pololei ʻole e hoʻolaha i ka holomua o ka tumor a hiki ke kāohi i nā pane pale endogenous. ʻO ka wehewehe hou aku o ke kuleana hana o kēia mau metabolites hoʻoponopono e wehe i nā hoʻolālā ʻē aʻe no ka hoʻoikaika ʻana i ka pane pale anti-tumor.
Ua loaʻa nā hāpana maʻi a me nā ʻikepili lapaʻau ma o ka waihona ʻiʻo puʻupuʻu kanesa BC i hōʻoia ʻia e ka Canadian Tissue Repository Network. Wahi a ke kaʻina hana i ʻāpono ʻia e ke Komite Hoʻoponopono Noiʻi Kanesa BC a me ke Kulanui o British Columbia (H07-00463), ua loaʻa i nā hāpana maʻi a pau a me nā ʻikepili lapaʻau i ka ʻae palapala i kākau ʻia a i ʻole ua hōʻole kūhelu i kā lākou ʻae. Mālama ʻia nā hāpana ma ka BioBank i hōʻoia ʻia (BRC-00290). Hōʻike ʻia nā ʻano kikoʻī o ka maʻi ma nā Papa S1 a me S5. No ka cryopreservation, hoʻohana ʻia kahi scalpel e hoʻokaʻawale i ka hāpana puʻupuʻu o ka mea maʻi a laila e hoʻokuke iā ia ma o kahi kānana 100-micron e loaʻa ai kahi hoʻokuʻu cell hoʻokahi. Ua centrifuged ʻia nā ascites o ka mea maʻi ma 1500 rpm no 10 mau minuke ma 4°C e pellet i nā cell a wehe i ka supernatant. Ua hoʻopaʻa ʻia nā pūnaewele i loaʻa mai ka puʻupuʻu a me nā ascites i loko o 50% serum AB kanaka i hoʻopau ʻia i ka wela (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) a me 10% dimethyl sulfoxide. Ua hoʻoheheʻe ʻia kēia mau hoʻokuʻu pūnaewele hoʻokahi i mālama ʻia a hoʻohana ʻia no ka metabolomics a me ka hoʻoholo ʻana i ka metabolite i wehewehe ʻia ma lalo nei.
ʻO ka medium piha he 0.22 μm kānana 50:50 i hoʻohui ʻia me RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) i hoʻohui ʻia me 10% serum kanaka AB i hoʻopau ʻia i ka wela (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Penicillin Streptomycin (PenStrep) solution (Thermo Fisher Scientific) a me 50 μMB-mercaptoethanol. Hoʻohui ʻia ʻo AimV (Invitrogen) me 20 mM Hepes (Thermo Fisher Scientific) a me 2 mM l-glutamine (Thermo Fisher Scientific). ʻO ka buffer staining cytometer flow he 0.22μm saline phosphate buffered i kānana ʻia (PBS; Invitrogen) i hoʻohui ʻia me 3% serum kanaka AB i hoʻopau ʻia i ka wela (Sigma). Ua haku ʻia ka buffer hoʻonui cell me 0.22μm PBS i kānana ʻia a hoʻohui ʻia me 0.5% serum kanaka AB i hoʻopau ʻia i ka wela (Sigma-Aldrich).
I loko o ka medium piha 37°C, ua hoʻoluʻu ʻia nā pūnaewele me 10 nM MT DR a me 100 μM 2-NBDG no 30 mau minuke. A laila, ua hoʻoluʻu ʻia nā pūnaewele me ka viability dye eF506 ma 4°C no 15 mau minuke. E hoʻokuʻu hou i nā pūnaewele ma FC Block (eBioscience) a me Brilliant Stain Buffer (BD Biosciences), e hoʻoheheʻe i loko o ka flow cytometer staining buffer (e like me nā kuhikuhi a ka mea hana), a hoʻomoʻa no 10 mau minuke ma ka mahana o ka lumi. E hoʻoluʻu i nā pūnaewele me kahi set o nā antibodies (Papa S2) i loko o ka flow cytometry staining buffer ma 4°C no 20 mau minuke. E hoʻokuʻu hou i nā pūnaewele i loko o ka flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) ma mua o ka nānā ʻana. E hoʻohana iā SpectroFlo a me FlowJo V10 e kālailai i ka ʻikepili helu kelepona, a e hoʻohana iā GraphPad Prism 8 e hana i ka ʻikepili. Ua hoʻonohonoho ʻia ka median fluorescence intensity (MFI) o 2-NBDG a me MT DR ma ke ʻano log-normalized, a laila ua hoʻohana ʻia kahi hoʻāʻo t i hui pū ʻia no ka loiloi helu e helu ai i nā maʻi i kūlike. Wehe i nā heluna kanaka āpau me ka emi ma mua o 40 mau hanana mai ka loiloi; e hoʻokomo i kahi waiwai MFI o 1 no kekahi mau waiwai maikaʻi ʻole ma mua o ka hana ʻana i ka loiloi helu a me ka nānā ʻana i ka ʻikepili.
I mea e hoʻohui ai i ka hoʻolālā gating manual o ka panela hana ma luna, ua hoʻohana mākou i ka annotation piha e ka lāʻau hoʻopaʻa ʻano (FAUST) (21) e hāʻawi aunoa i nā cell i ka heluna kanaka ma hope o ka hoʻopau ʻana i nā cell make ma FlowJo. Hoʻokele lima mākou i ka hopena e hoʻohui i nā heluna kanaka i ʻike ʻia ua hewa ka hoʻokaʻawale ʻana (e hoʻohui ana iā PD1+ me nā cell PD1-tumor) a me nā heluna kanaka i mālama ʻia. Loaʻa i kēlā me kēia laʻana ka awelika o nā cell ma mua o 2%, no ka huina o 11 mau heluna kanaka.
Ua hoʻohana ʻia ka Ficoll gradient density centrifugation e hoʻokaʻawale i ka PBMC mai nā huahana hoʻokaʻawale leukocyte (STEMCELL Technologies). Ua hoʻokaʻawale ʻia nā pūnaewele CD8 + T mai PBMC me ka hoʻohana ʻana i ka CD8 MicroBeads (Miltenyi) a hoʻonui ʻia i loko o ka medium piha me ka hoʻohana ʻana iā TransAct (Miltenyi) no 2 mau pule e like me nā kuhikuhi a ka mea hana. Ua ʻae ʻia nā pūnaewele e kū no 5 mau lā i loko o ka medium piha e loaʻa ana iā IL-7 (10 ng/ml; PeproTech), a laila hoʻoulu hou ʻia me TransAct. I ka lā 7, e like me nā kuhikuhi a ka mea hana, ua hoʻohana ʻia nā CD45 MicroBeads kanaka (Miltenyi) e hoʻonui i nā pūnaewele i ʻekolu mau puni. Ua hoʻokaʻawale ʻia nā pūnaewele no ka nānā ʻana i ka cytometry kahe (e like me ka mea i wehewehe ʻia ma luna), a ua hoʻokaʻawale ʻia hoʻokahi miliona mau pūnaewele i ʻekolu manawa no ka nānā ʻana o LC-MS/MS. Ua hana ʻia nā laʻana e LC-MS/MS e like me ka mea i wehewehe ʻia ma lalo nei. Ua kuhi mākou i ka waiwai metabolite i nalowale me ka helu ion o 1,000. Hoʻonohonoho ʻia kēlā me kēia hāpana e ka helu ion holoʻokoʻa (TIC), i hoʻololi ʻia ma ke ʻano logarithmically a hoʻonohonoho ʻia maʻamau ma MetaboAnalystR ma mua o ka nānā ʻana.
Ua hoʻoheheʻe ʻia ka hoʻokuʻu ʻana o nā cell hoʻokahi o kēlā me kēia maʻi a kānana ʻia ma o kahi kānana 40 μm i loko o ka medium piha (e like me ka mea i wehewehe ʻia ma luna). Wahi a ke kaʻina hana a ka mea hana, ʻekolu mau pōʻai koho maikaʻi ma o ka hoʻokaʻawale ʻana o nā bead magnetic me ka hoʻohana ʻana iā MicroBeads (Miltenyi) i hoʻohana ʻia e hoʻonui i nā laʻana no nā cell CD8+, CD4+ a me CD45- (ma luna o ka hau). I ka pōkole, ua hoʻokuʻu hou ʻia nā cell i loko o ka buffer hoʻonui cell (e like me ka mea i wehewehe ʻia ma luna) a helu ʻia. Ua hoʻoulu ʻia nā cell me nā beads CD8 kanaka, nā beads CD4 kanaka a i ʻole nā ​​beads CD45 kanaka (Miltenyi) ma 4°C no 15 mau minuke, a laila holoi ʻia me ka buffer hoʻonui cell. Hoʻouna ʻia ka laʻana ma o ke kolamu LS (Miltenyi), a ua hōʻiliʻili ʻia nā ʻāpana maikaʻi a me nā ʻāpana maikaʻi ʻole. I mea e hōʻemi ai i ka lōʻihi a hoʻonui i ke kaʻina hoʻōla cell, a laila hoʻohana ʻia ka CD8-fraction no ka lua o ka pōʻai o ka hoʻonui ʻana o CD4+, a hoʻohana ʻia ka CD4-fraction no ka hoʻonui ʻana o CD45 ma hope. E mālama i ka hopena ma luna o ka hau ma ke kaʻina hana hoʻokaʻawale.
No ka hoʻomākaukau ʻana i nā laʻana no ka nānā ʻana i ka metabolite, ua holoi ʻia nā ʻāpana i hoʻokahi manawa me ka wai paʻakai anu, a ua hoʻohui ʻia he 1 ml o 80% methanol i kēlā me kēia laʻana, a laila vortexed a snap hau i loko o ka naikokene wai. Ua hoʻokau ʻia nā laʻana i ʻekolu mau pōʻaiapuni hau-hoʻoheheʻe a centrifuged ma 14,000 rpm no 15 mau minuke ma 4°C. Hoʻoheheʻe ʻia ka supernatant e loaʻa ana nā metabolites a maloʻo. Ua hoʻoheheʻe hou ʻia nā metabolites i loko o 50 μl o 0.03% formic acid, vortexed e hui pū, a laila centrifuged e wehe i nā ʻōpala.
E unuhi i nā metabolites e like me ka mea i wehewehe ʻia ma luna. E hoʻololi i ka supernatant i kahi ʻōmole chromatography wai hana kiʻekiʻe no ka noiʻi metabolomics. E hoʻohana i kahi protocol lapaʻau random e mālama i kēlā me kēia laʻana me ka helu like o nā cell e pale aku i nā hopena batch. Ua hana mākou i kahi loiloi qualitative o nā metabolites honua i paʻi mua ʻia ma ka AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Ua hana ʻia ka nānā ʻana o Chromatographic a me ka hoʻohui ʻana o ka peak area me ka hoʻohana ʻana i ka polokalamu MultiQuant mana 2.1 (Applied Biosystems SCIEX).
Ua hoʻohana ʻia kahi helu ion o 1000 e kuhi i ka waiwai metabolite i nalowale, a ua hoʻohana ʻia ka TIC o kēlā me kēia laʻana e helu i ka wahi piko maʻamau o kēlā me kēia metabolite i ʻike ʻia e hoʻoponopono i nā loli i hoʻolauna ʻia e ka loiloi instrumental mai ka hana ʻana i ka laʻana. Ma hope o ka maʻamau ʻana o TIC, hoʻohana ʻia ʻo MetaboAnalystR(51) (paʻamau paʻamau) no ka hoʻololi logarithmic a me ka scaling laina maʻamau aunoa. Ua hoʻohana mākou iā PCA me ka pūʻolo vegan R e hana i ka loiloi ʻimi o nā ʻokoʻa metabolome ma waena o nā ʻano laʻana, a hoʻohana i ka loiloi redundancy hapa e kālailai i nā maʻi. E hoʻohana i ke ʻano Ward e kūkulu i kahi dendrogram palapala wela e hui pū i ka mamao Euclidean ma waena o nā laʻana. Ua hoʻohana mākou iā limma (52) ma ka nui o ka metabolite maʻamau e ʻike i nā metabolites nui like ʻole ma ke ʻano cell holoʻokoʻa a me ka microenvironment. I mea e hoʻomaʻalahi ai i ka wehewehe ʻana, hoʻohana mākou i ka parameter mean group e kuhikuhi i ke kumu hoʻohālike, a noʻonoʻo i nā ʻano cell i loko o ka microenvironment e like me kēlā me kēia hui (n = 6 mau hui); no ka hoʻāʻo koʻikoʻi, ua hana mākou i ʻekolu mau ana hou no kēlā me kēia metabolite I mea e pale aku ai i ka hana hou ʻana, ua hoʻokomo ʻia ka mea maʻi ma ke ʻano he keʻakeʻa i ka hoʻolālā limma. I mea e nānā ai i nā ʻokoʻa o nā metabolites ma waena o nā maʻi like ʻole, ua hoʻoponopono mākou i ke kumu hoʻohālike limma me nā maʻi ma ke ʻano paʻa. Hōʻike mākou i ke koʻikoʻi o ka ʻokoʻa i kuhikuhi mua ʻia ma waena o ke ʻano cell a me ka microenvironment o Padj <0.05 (hoʻoponopono Benjamini-Hochberg).
Ma hope o ka hoʻonui ʻana i ka ikaika me ka hoʻohana ʻana iā Miltenyi Dead Cell Removal Kit (>80% viability), ua hana ʻia ke kaʻina hana transcriptome hoʻokahi-cell ma nā ascites hau ola a me nā laʻana tumor me ka hoʻohana ʻana i kahi protocol expression 10x 5′gene. Ua kālailai ʻia ʻelima mau hihia me nā tumors kūlike a me nā ascites, ʻoiai ʻo ka viability haʻahaʻa mai hoʻokahi laʻana tumor i pale i kona hoʻokomo ʻia ʻana. I mea e hoʻokō ai i nā koho he nui o nā maʻi, ua hoʻohui mākou i nā laʻana o kēlā me kēia maʻi ma nā ala o ka mea hoʻokele chromium 10x, a kālailai i nā ascites a me nā wahi tumor ma ke kaʻawale. Ma hope o ke kaʻina hana [Illumina HiSeq 4000 28 × 98 bp paired end (PE), Quebec genome; ʻo ka awelika o 73,488 a me 41,378 heluhelu no kēlā me kēia cell no ka tumor a me ka ascites]], ua hoʻohana mākou iā CellSNP a me Vireo (53) (ma muli o CellSNP e like me Ua hāʻawi ʻia ka SNP kanaka maʻamau (VCF) i hāʻawi ʻia e GRCh38 i kahi ʻike donor. Hoʻohana mākou iā SNPRelate e hoʻoholo i ka ʻike kokoke loa (IBS) o ke kūlana genotype o ka mea maʻi (IBS), e kāpae ana i nā cell i hāʻawi ʻole ʻia a me nā cell i ʻike ʻia he duplexes a me nā mea hāʻawi like ma waena o ascites a me nā laʻana tumor (54). Ma ke kumu o kēia hana, ua mālama mākou i ʻekolu mau hihia me ka hōʻike cell nui i loko o ka tumor a me nā ascites no ka nānā ʻana ma hope. Ma hope o ka hana ʻana i kahi ʻanuʻu kānana nui i loko o ka scater (55) a me ka scran (56) BioConductor packaging, ua loaʻa kēia i 6975 mau cell (2792 a me 4183 mau cell mai ka tumor a me ka ascites, kēlā me kēia) no ka nānā ʻana. Hoʻohana mākou iā igraph's (57) Louvain clustering o ka pūnaewele hoalauna kokoke loa (SNN) ma muli o ka mamao Jaccard i nā cell cluster ma o ka hōʻike ʻana. Ua hoʻopaʻa lima ʻia nā hui i nā ʻano cell putative e pili ana i ka hōʻike ʻana o ka gene marker a ua nānā ʻia me t-SNE. Ua wehewehe ʻia nā cell T Cytotoxic e ka hōʻike ʻana o CD8A a me GZMA, koe wale nō nā subclusters me ka hōʻike protein ribosomal haʻahaʻa. Ua komo mākou i ka ʻikepili i paʻi ʻia o Izar et al. (16), me kā lākou hoʻokomo ʻana o t-SNE, hiki ke kaohi i ka overlap expression ma waena o nā māka cell immune a me ka hōʻike ʻana o NNMT.
Ua hoʻokaʻawale ʻia ʻo PBMC mai nā huahana hoʻokaʻawale leukocyte (STEMCELL Technologies) e ka Ficoll gradient density centrifugation. Ua hoʻokaʻawale ʻia nā pūnaewele CD3 + mai PBMC me ka hoʻohana ʻana i nā beads CD3 (Miltenyi). I ke alo a i ʻole ka ʻole o MNA, ua hoʻāla ʻia nā pūnaewele CD3 + me ka plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) a me IL-2 (300 U/ml; Proleukin). I ka lā hope o ka hoʻonui ʻana, ua loiloi ʻia ke ola (Fixable Viability Dye eFluor450, eBioscience) a me ka proliferation (123count eBeads, Thermo Fisher Scientific) e ka flow cytometry. E loiloi i ka hana effector ma ka hoʻoulu ʻana i nā cell me PMA (20 ng/ml) a me ionomycin (1μg/ml) me GolgiStop no 4 mau hola, a nānā iā CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) a me TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). E hoʻoulu i nā cell qPCR a me ChIP me PMA (20 ng/ml) a me ionomycin (1μg/ml) no 4 mau hola. Ua hōʻiliʻili ʻia ka supernatant ELISA ma mua a ma hope o ka hoʻoulu ʻana me PMA (20 ng/ml) a me ionomycin (1 μg/ml) no 4 mau hola.
E hahai i ke kaʻina hana a ka mea hana e hoʻokaʻawale i ka RNA me ka hoʻohana ʻana iā RNeasy Plus Mini Kit (QIAGEN). E hoʻohana iā QIAshredder (QIAGEN) e hoʻohui i ka laʻana. E hoʻohana i ka RNA kiʻekiʻe-mana i ka pahu cDNA (Thermo Fisher Scientific) e hana i ka DNA hoʻopihapiha (cDNA). E hoʻohana iā TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) e helu i ka hōʻike ʻana o ka gene (e like me ke kaʻina hana a ka mea hana) me kēia mau probes: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] a me Hs01010726_m1 (SLC22A2). Ua holo ʻia nā laʻana ma ka ʻōnaehana PCR manawa maoli ʻo StepOnePlus (Applied Biosystems) (Applied Biosystems) ma ka papa hana optical 96-well MicroAmp wikiwiki (Applied Biosystems) me ka ʻili optical MicroAmp. ʻO kēlā me kēia waiwai Ct e ʻoi aku ma mua o 35 ua manaʻo ʻia he ʻoi aku ma mua o ka paepae ʻike a ua hōʻailona ʻia he hiki ʻole ke ʻike ʻia.
E hana i ka ChIP e like me ka mea i wehewehe mua ʻia (58). I ka pōkole, ua mālama ʻia nā cell me ka formaldehyde (ka hoʻohui hope loa 1.42%) a ua hoʻomoʻa ʻia ma ka mahana o ka lumi no 10 mau minuke. E hoʻohana i ka buffer pehu i hoʻohui ʻia (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl a me 0.1% NP-40) ma luna o ka hau no 10 mau minuke, a laila hoʻomoʻa hou i loko o ka buffer immunoprecipitation e like me ka mea i wehewehe ʻia (58). A laila ua sonicated ʻia ka laʻana me nā pōʻaiapuni aʻe: 10 mau pōʻaiapuni (20 pulses 1-kekona) a me ka manawa static o 40 kekona. E hoʻomoʻa i ka ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) a me SP1 (Cell Signaling Technology; 3μl) antibodies me ka laʻana ma 4°CC lulu i ka pō. E hoʻomoʻa i nā beads protein A (Thermo Fisher Scientific) me ka hāpana ma 4°C me ka lulu mālie no 1 hola, a laila e hoʻohana i nā beads chelex (Bio-Rad) e hoʻonui i ka DNA, a hoʻohana i ka proteinase K (Thermo Fisher) no ka ʻeli ʻana o ka protein. Ua ʻike ʻia ka mea hoʻolaha TNFα e PCR: forward, GGG TAT CCT TGA TGC TTG TGT; ma ke ʻano ʻē, GTG CCA ACA ACT GCC TTT ATA TG (huahana 207-bp). Ua hana ʻia nā kiʻi e Image Lab (Bio-Rad) a ua helu ʻia me ka hoʻohana ʻana i ka polokalamu ImageJ.
Ua hōʻiliʻili ʻia ka supernatant moʻomeheu cell e like me ka mea i wehewehe ʻia ma luna. Ua hoʻokō ʻia ka hoʻoholo ʻana e like me nā kaʻina hana a ka mea hana o ka pahu TNFα ELISA kanaka (Invitrogen), pahu IL-2 ELISA kanaka (Invitrogen) a me ka pahu IFN-γ ELISA kanaka (Abcam). Wahi a ke kaʻina hana a ka mea hana, ua hoʻoheheʻe ʻia ka supernatant 1:100 e ʻike iā TNFα a me IL-2, a me 1:3 e ʻike iā IFN-γ. E hoʻohana iā EnVision 2104 Multilabel Reader (PerkinElmer) e ana i ka absorbance ma 450 nm.
Ua hoʻokaʻawale ʻia ʻo PBMC mai nā huahana hoʻokaʻawale leukocyte (STEMCELL Technologies) e ka Ficoll gradient density centrifugation. Ua hoʻokaʻawale ʻia nā pūnaewele CD3 + mai PBMC me ka hoʻohana ʻana i nā beads CD3 (Miltenyi). I ke alo a i ʻole ka ʻole o MNA, ua hoʻāla ʻia nā pūnaewele CD3 + me ka plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) a me IL-2 (300 U/ml; Proleukin) no 3 mau lā. Ma hope o 3 mau lā, ua hōʻiliʻili ʻia nā pūnaewele a holoi ʻia me ka 0.9% saline, a ua hoʻopaʻa ʻia ka pellet. Ua hana ʻia ka helu ʻana o nā pūnaewele ma o ka flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) me ka hoʻohana ʻana i ka 123count eBeads.
E unuhi i nā metabolites e like me ka mea i wehewehe ʻia ma luna. Ua hoʻohui hou ʻia ka extract maloʻo ma kahi nui o 4000 cell equivalents/μl. E kālailai i ka laʻana ma o ka reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) a me ke kolamu CORTECS T3 (2.1 × 150 mm, ka nui o ka ʻāpana 1.6-μm, ka nui o ka pore 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), kahi e hana ai ka electrospray ionization ma ke ʻano maikaʻi. ʻO ka pae kelepona A he 0.1% formic acid (ma H2O), ʻo ka pae kelepona B he 90% acetonitrile, 0.1% formic acid. ʻO ke kahe LC he 0 a 2 mau minuke no 100% A, 2 a 7.1 mau minuke no 99% B, a me 7.1 a 8 mau minuke no 99% B. A laila e hoʻoponopono hou i ke kolamu me ka pae lawe A ma ka nui o ke kahe ʻana o 0.6 ml/min no 3 mau minuke. . ʻO ka nui o ke kahe ʻana he 0.4ml/min, a ua hoʻomehana ʻia ke keʻena kolamu i 50°C. E hoʻohana i ke kūlana kemika maʻemaʻe o MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Kanada) e hoʻokumu i ka manawa paʻa (RT) a me ka hoʻololi ʻana (RT = 0.882 mau minuke, hoʻololi 1 = 137→94.1, hoʻololi 2 = 137→92, Hoʻololi 3 = 137→78). Ke hana ʻia nā hoʻololi ʻekolu i ka manawa paʻa kūpono, hoʻohana ʻia ka hoʻololi 1 no ka helu ʻana e hōʻoia i ka kikoʻī. Ua hoʻokumu ʻia ke kiʻikuhi maʻamau o MNA (Toronto Research Chemical Company) e ʻeono mau dilutions serial o ka hopena waihona (1 mg/ml) e loaʻa ai nā kūlana o 0.1, 1.0, 10 a me 100 ng/ml a me 1.0 a me 10μg/ml wai. ʻO ka palena ʻike he 1 ng/ml, a ʻo ka pane linear ma waena o 10 ng/ml a me 10μg/ml. Hoʻohana ʻia kēlā me kēia injection o ʻelua microliters o ka laʻana a me ke kūlana no ka loiloi LC/MS, a holo ʻia kahi laʻana kaohi maikaʻi i hui ʻia i kēlā me kēia ʻewalu injections e hōʻoia i ke kūpaʻa o ka paepae loiloi. Aia nā pane MNA o nā laʻana cell i mālama ʻia me MNA i loko o ka laulā linear o ka assay. Ua hana ʻia ka loiloi ʻikepili me ka hoʻohana ʻana i ka polokalamu loiloi quantitative MassHunter (v9.0, Agilent).
Ua lawe ʻia ke kūkulu ʻana o ka hanauna ʻelua o αFR-CAR mai Song et al. (59). I ka pōkole, aia i loko o ke kūkulu nā ʻike penei: kaʻina alakaʻi CD8a, ka ʻāpana loli kaulahao hoʻokahi kikoʻī o ke kanaka αFR, ka ʻāpana hinge CD8a a me ka transmembrane, ka ʻāpana intracellular CD27 a me ka ʻāpana intracellular CD3z. Ua hoʻohui ʻia ke kaʻina CAR piha e GenScript, a laila cloned i loko o ka vector hōʻike lentiviral hanauna ʻelua ma luna o ka cassette hōʻike GFP i hoʻohana ʻia e loiloi i ka pono o ka transduction.
Hoʻopuka ʻia ʻo Lentivirus ma o ka transfection o nā pūnaewele HEK293T [American Type Culture Collection (ATCC); i ulu ʻia ma ka Dulbecco's modified Eagle medium e loaʻa ana he 10% fetal bovine serum (FBS) a me 1% PenStrep, a hoʻohana ʻia ka CAR-GFP vector a me nā plasmids packaging (psPAX2 a me pMD2.G, Addgene) e hoʻohana i ka lipofection amine (Sigma-Aldrich). Ua hōʻiliʻili ʻia ka supernatant i loaʻa ka virus he 48 a me 72 mau hola ma hope o ka transfection, kānana ʻia, a hoʻohuihui ʻia e ka ultracentrifugation. E mālama i ka supernatant viral i hoʻohuihui ʻia ma -80°C a hiki i ka transduction.
Ua hoʻokaʻawale ʻia nā PBMC mai nā huahana hoʻokaʻawale leukocyte donor olakino (STEMCELL Technologies) e Ficoll gradient density centrifugation. E hoʻohana i nā microbeads CD8 koho maikaʻi (Miltenyi) e hoʻokaʻawale i nā pūnaewele CD8+ mai PBMC. E hoʻoulu i nā pūnaewele T me TransAct (Miltenyi) a ma ka medium TexMACS [Miltenyi; i hoʻohui ʻia me 3% serum kanaka i hoʻopau ʻia i ka wela, 1% PenStrep a me IL-2 (300 U/ml)]. Iwakāluakūmāhā hola ma hope o ka hoʻoulu ʻana, ua hoʻololi ʻia nā pūnaewele T me ka lentivirus (10 μl supernatant virus i hoʻohui ʻia no 106 mau pūnaewele). 1 a 3 mau lā ma hope o ka hoʻololi ʻana ma Cytek Aurora (ma FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), e loiloi i ka hōʻike ʻana o GFP o nā pūnaewele e hōʻike i ka pono o ka hoʻololi ʻana ma ka liʻiliʻi he 30%.
Ua hoʻoulu ʻia nā pūnaewele CAR-T no 24 mau hola ma Immunocult (STEMCELL Technologies; i hoʻohui ʻia me 1% PenStrep) ma lalo o nā kūlana penei: ʻaʻole i mālama ʻia, mālama ʻia me 250 μM adenosine a i ʻole 10 mM MNA. Ma hope o ka mālama mua ʻana, ua holoi ʻia nā pūnaewele CAR-T me PBS a hui pū ʻia me 20,000 mau pūnaewele SK-OV-3 [ATCC; ma ka McCoy 5A medium (Sigma-Aldrich) i hoʻohui ʻia me 10% FBS a me 1% PenStrep ma 10: Ua hoʻonui ʻia ka ratio effector i ka target o 1 ma triplicate i ka Immunocult medium i hoʻohui ʻia. Ua hoʻohana ʻia nā pūnaewele SK-OV-3 a me nā pūnaewele SK-OV-3 i hoʻoheheʻe ʻia me ka digitalis saponin (0.5mg/ml; Sigma-Aldrich) ma ke ʻano he mau kaohi maikaʻi ʻole a maikaʻi hoʻi. Ma hope o 24 mau hola o ka mahi ʻana like, ua hōʻiliʻili ʻia ka supernatant a ua ana ʻia ka lactate dehydrogenase (LDH) e like me nā kuhikuhi a ka mea hana (LDH Glo Cytotoxicity Assay Kit, Promega). Ua hoʻoheheʻe ʻia ka supernatant LDH 1:50 i loko o ka buffer LDH. Ua ana ʻia ka pakeneka o ka pepehi ʻana me ka hoʻohana ʻana i kēia ʻano hana: pakeneka o ka pepehi ʻana = pakeneka o ka hoʻoponopono / ka nui o ka pepehi ʻana x 100%, kahi o ka pakeneka o ka hoʻoponopono = nā pūnaewele T co-culture-wale nō, a me ka nui o ka pepehi ʻana = ka mana maikaʻi-ka mana maikaʻi ʻole.
E like me ka mea i wehewehe ʻia ma ke kikokikona a i ʻole nā ​​​​mea a me nā ʻano hana, e hoʻohana i ka GraphPad Prism 8, Microsoft Excel a i ʻole R v3.6.0 no ka loiloi helu. Inā hōʻiliʻili ʻia nā laʻana he nui mai ka mea maʻi hoʻokahi (e like me ka ascites a me ka tumor), hoʻohana mākou i kahi hoʻāʻo t i hui pū ʻia a hoʻokomo paha i ka mea maʻi ma ke ʻano he hopena random i loko o kahi kumu hoʻohālike linear a i ʻole generalized e like me ka mea kūpono. No ka loiloi metabolomics, hana ʻia ka hoʻāʻo koʻikoʻi ma triplicate.
No nā mea kōkua no kēia ʻatikala, e ʻoluʻolu e nānā i 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. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. Jones), De Bellardini (Ralph J. Jones), De Bellardini (Ralph J. Jones) Phineas T. Hamilton (Phineas T.
Hāʻawi ka MNA i ka hoʻopau ʻana i nā pūnaewele T a ke hōʻike nei i kahi pahuhopu immunotherapy kūpono no ka mālama ʻana i ka maʻi kanesa kanaka.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. Jones), De Bellardini (Ralph J. Jones), De Bellardini (Ralph J. Jones) Phineas T. Hamilton (Phineas T.
Hāʻawi ka MNA i ka hoʻopau ʻana i nā pūnaewele T a ke hōʻike nei i kahi pahuhopu immunotherapy kūpono no ka mālama ʻana i ka maʻi kanesa kanaka.
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