Banakisɛfagalanw ye banakisɛfagalanw ye minnu bɛ kɛ tumuw lamini na (TME), nka ni danfara damadɔw tɛ, u dɔnni fanba ma dɔn. Nin yɔrɔ in na, an ye kurukuruw ni T-cellw sɛgɛsɛgɛ ka bɔ banabagatɔw ka kurukuruw ni u ka sɔgɔsɔgɔninjɛ la minnu ka kansɛri jugumanba (HGSC) bɛ u la walasa ka nin TME yɔrɔ suguya ninnu ka metabolome jira. Asiti ni tumokisɛw bɛ danfaraba don farikolojɔlifɛnw na. Ni aw ye a suma ni asidi ye, T-cell minnu bɛ don tumu kɔnɔ, olu nafa ka bon kosɛbɛ 1-methylnicotinamide (MNA) la. Hali ni MNA hakɛ bɛ T-cellw la, nikotinamidi N-methyltransferase (enzyme min bɛ metil kuluw jiginni kɛ ka bɔ S-adenosylmethionine la ka taa nikotinamidi la) jirali bɛ dan fibroblaste ni tumor cellules ma. Baarakɛcogo la, MNA bɛ T-cellw bila ka sitokini (cytokine tumor necrosis factor alpha) bɔ min bɛ tumo yiriwa. O la, MNA min bɛ bɔ TME la, o bɛ dɛmɛ don T-cellw ka farikolo tangacogo la, wa a bɛ kɛ farikolo tangacogo laɲini ye min bɛ se ka kɛ hadamadenw ka kansɛri furakɛli la.
Tumu minnu bɛ bɔ tumu na, olu bɛ se ka nɔba bila tumu kɛlɛli farikolo tangacogo la, wa dalilu caman b’a jira ko u bɛ se ka kɛ fana fanga jɔnjɔn ye bana ka taa ɲɛ (1). Ka fara Warburg nɔ kan, kɔsa in na baara daminɛna walasa ka tumokisɛw ka farikoloɲɛnajɛ cogoya jira ani a ni tumuw ka sigida farikolo tangacogo (TME) cɛsira. Kɔlɔsili minnu kɛra sosow ka misaliw ni hadamadenw ka T-cellw kan, olu y’a jira ko glutamine farikoloɲɛnajɛ (2), oksidan sɛnɛcogo (3) ani sukaro sɛnɛcogo (4) bɛ se ka baara kɛ u yɛrɛma farikolo tangalanw kulu fitinin suguya caman kan. O siraw la, farikolojɔlifɛn caman bɛ T-cellw ka tumu kɛlɛli baara bali. A jirala ko tetarasikilini (BH4) coenzyme balili bɛ se ka T-cell caya tiɲɛ, wa ni BH4 caya farikolo la, o bɛ se ka farikolo tangacogo fanga bonya min bɛ sɔrɔ CD4 ni CD8 fɛ. Ka fara o kan, kynurenine ka farikolo tangalan nɔ bɛ se ka kisi ni BH4 tali ye (5). Isocitrate dehydrogenase (IDH) mutant glioblastoma kɔnɔ, enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) bɔli bɛ T selilɛriw ka baarakɛcogo, u caya ani u ka sitolisi baara bali (6). Kɔsa in na, a jirala ko metilgiliyɔkisali, n’o ye glycolyse kɔfɛfɛn ye, o bɛ sɔrɔ suppresseur cellules fɛ minnu bɔra myeloïde la, wa T cell transfer of methylglyoxal bɛ se ka effecteur T cell baara bali. Furakɛli la, metilgiliyɔkisali neutralisation bɛ se ka se sɔrɔ myeloïde-derived suppressor cells (MDSC) ka baara kan ani ka ɲɔgɔndɛmɛ ka checkpoint blockade furakɛli bonya sosow ka misaliw la (7). Nin kalan ninnu faralen ɲɔgɔn kan, u bɛ sinsin TME-sɔrɔ-fɛnw jɔyɔrɔba kan T-cell baarakɛcogo n’u ka baarakɛcogo ɲɛnabɔli la.
T-cell baarakɛbaliya kofɔra kosɛbɛ kɔnɔbara kansɛri la (8). O bɛ bɔ farikolojɔli jogo dɔw la minnu bɛ sɔrɔ oksizɛni dɔgɔyali ni tumu joli siraw la minnu tɛ cogo la (9), o bɛ na ni sukaro ni triptofan jiginni ye ka kɛ fɛn wɛrɛw ye i n’a fɔ asidi laktiki ni kinurenini. Laktati kɔkanna tɛmɛnen bɛ dɔ bɔ interferon-γ (IFN-γ) sɔrɔli la ani ka miyɛlosuppressive kulu fitininw danfara bila sen kan (10, 11). Triptophane dunni bɛ T-cell caya bali k’a ɲɛsin a yɛrɛ ma ani ka T-cell minɛbaga taamasiɲɛw bali (12-14). Nin kɔlɔsili ninnu bɛɛ n’a ta, baara caman kɛra farikolo tangacogo lamini na in vitro T cell culture (in vitro) la ni kunnafonidilanw ye minnu bɛ se ka kɛ, walima minnu dan ye sosow ka misali kelenw ye in vivo, o fila si tɛ hadamadenw ka kansɛriw ni Physiological macro and micro environment (Fysiological macro and micro environment) danfara jira bɛrɛbɛrɛ.
Kɔnɔbara kansɛri taamasiɲɛ min ka teli ka sɔrɔ, o ye kɔnɔbara basigilen jɛnsɛnni ye ani sɔgɔsɔgɔninjɛ bɔli. Kɔnɔnatumuw ka ji dalajɛli asidi la, o bɛ tali kɛ bana jugumanba la ani a ɲɛfɔcogo jugu la (15). Jateminɛw y’a jira ko nin yɔrɔ kɛrɛnkɛrɛnnen in ye oksizɛni dɔgɔyali ye, joli siraw falenfalenni (VEGF) ni indoleamine 2,3-dioxygenase (IDO) hakɛ ka ca a la, wa T ɲɛnabɔli selilɛriw ni miyɛloyi balilanw bɛ don a kɔnɔ (15-18). Asiti farikoloɲɛnajɛ sigida bɛ se ka kɛ danfara ye ni kuru yɛrɛ ta ye, o la T-cellw ka porogaramu seginni kɔnɔbara yɔrɔ la, o ma jɛya. Ka fara o kan, danfaraba minnu bɛ asiti ni farikolojɔlifɛnw cɛ minnu bɛ tumu lamini na, olu bɛ se ka farikolo tangalanw doncogo bali ani u ka baara kɛ tumuw kan, wa ɲinini wɛrɛw ka kan ka kɛ.
Walasa ka nin gɛlɛya ninnu ɲɛnabɔ, an ye sensitive cell separation and liquid chromatography tandem mass spectrometry (LC-MS/MS) fɛɛrɛ dɔ labɛn walasa ka selilu suguya wɛrɛw sɛgɛsɛgɛ (CD4 + ni CD8 + T cellules fana sen bɛ o la) ka fara tumuw kɔnɔ ani u ni ɲɔgɔn cɛ A farikolojɔlifɛnw bɛ seliluw span ascite ni banabagatɔ ka tumo sigida kelen na. An bɛ baara kɛ ni nin fɛɛrɛ in ye ka fara flow cytometry high-dimensional ani single-cell ARN sequencing (scRNA-seq) kan walasa ka ja dɔ di min ɲɛnabɔlen don kosɛbɛ nin jamanaden jɔnjɔn ninnu ka farikoloɲɛnajɛ cogoya kan. Nin fɛɛrɛ in y’a jira ko dɔ farala kosɛbɛ 1-methylnicotinamide (MNA) hakɛ kan tumu T selilɛriw la, wa, sɛgɛsɛgɛli minnu kɛra in vitro la, olu y’a jira ko MNA ka farikolo tangacogo nɔ min bɛ T selilɛriw baara la, o tun ma dɔn fɔlɔ. A ka ca a la, nin fɛɛrɛ in bɛ ɲɔgɔn falenfalen jira tumuw ni farikolo tangalanw cɛ, wa a bɛ hakilina kɛrɛnkɛrɛnnenw di farikolo tangalanw ɲɛnabɔli farikolojɔlifɛnw kan, minnu bɛ se ka kɛ nafa ye T selilɛriw furakɛli la kɔnɔbara basigilen kansɛri furakɛli la Furakɛli cogoyaw.
An ye baara kɛ ni flow cytometry high-dimensional ye walasa ka sukaro tali hakɛ jateminɛ waati kelen na [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) ani mitokondriyaw ka baara [MitoTracker Deep Red (MT DR)] (7, 19, 20) bɛ kɛrɛfɛ cogoya la taamasiɲɛw minnu bɛ farikolo tangalanw ni tumuw hakɛ danfara bɔ (Taabolo S2 ani ja S1A). Nin sɛgɛsɛgɛli in y’a jira ko ni i ye a suma ni T-cell ye, ascite ni tumor cellules ka sukaro tali hakɛ ka ca, nka danfara ka dɔgɔn mitokondriyaw ka baara la. Tumukisɛw [CD45-EpCAM (EpCAM)+] ka sukaro tali hakɛ cayalenba ye T selilɛriw ta ye siɲɛ saba fo siɲɛ naani, ani CD4 + T selilɛriw ka sukaro tali hakɛ cayalenba ye CD8 + T selilɛriw ta hakɛ ye siɲɛ 1,2, o b’a jira ko Tumu infiltrating lymphocytes (TIL) ka farikoloɲɛnajɛ magow tɛ kelen ye hali TME kelen na (Ja 1A). O ni mitokondriyaw ka baara ta tɛ kelen ye tumuw la, o ni CD4 + T selilɛriw ta bɛ tali kɛ ɲɔgɔn na, wa farikolokisɛ suguya fila bɛɛ ka mitokondriyaw ka baara ka ca ni CD8 + T selilɛriw ta ye (Jaa 1B). A ka c’a la, nin jaabi ninnu bɛ farikoloɲɛnajɛ hakɛ jira. Tumukisɛw ka farikoloɲɛnajɛ baara ka ca ni CD4 + T selilɛriw ta ye, ani CD4 + T selilɛriw ka farikoloɲɛnajɛ baara ka ca ni CD8 + T selilɛriw ta ye. Hali ni nin nɔ ninnu bɛ se ka kɛ selilɛri suguya bɛɛ la, danfara basigilen tɛ CD4 + ni CD8 + T selilɛriw ka farikoloɲɛnajɛ cogoya la walima u hakɛ danfaralenw na sɔgɔsɔgɔninjɛ la ni i ye a suma ni tumuw ye (Jaa 1C). O ni CD45-cell fraction ta tɛ kelen ye, EpCAM+ selilɛriw hakɛ cayara tumu kɔnɔ ni i ye a suma ni asiti ye (Jaa 1D). An ye danfara jɛlen fana kɔlɔsi farikoloɲɛnajɛ la EpCAM+ ni EpCAM- selilɛri yɔrɔw cɛ. EpCAM+ (tumor) seliluw ka sukaro tali ni mitokondriyaw ka baara ka bon ka tɛmɛ EpCAM- selilɛriw kan, o ka ca kosɛbɛ ni fibroblastew ka farikoloɲɛnajɛ baara ye tumu selilɛriw kɔnɔ TME kɔnɔ (Jaa 1, E ani F).
. (C) CD4 + ni CD8 + selilɛriw (CD3 + T selilɛriw) hakɛ bɛ sɔrɔ asidi ni kuru la. (D) EpCAM + tumokisɛw hakɛ bɛ sɔrɔ asidi ni kuru la (CD45−). (E ni F) EpCAM + CD45-tumor ani EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) ani mitokondriyaw ka baara (MitoTracker bilen dibiman) (F) ja jiralenw (numan fɛ) ani kunnafoniw jatebɔlenw ( Niman fɛ) Asiti ni tumu selilɛriw. (G) CD25, CD137 ani PD1 jiracogo ja jiralenw ni flow cytometry ye. (H ni I) CD25, CD137 ani PD1 jirali CD4 + T selilɛriw kan (H) ani CD8 + T selilɛriw (I). (J ni K) Naive, central memory (Tcm), effector (Teff) ani effector memory (Tem) fenotipi minnu sinsinnen bɛ CCR7 ni CD45RO jirali kan. Ja minnu bɛ CD4 + T selilɛriw (J) ni CD8 + T selilɛriw (K) jira (numan fɛ) ani tabali kunnafoniw (niman fɛ) minnu bɛ asiti ni kuruw la. P hakɛw bɛ latigɛ ni t-test fila-fila ye (*P<0,05, **P<0,01 ani ***P<0,001). Layini bɛ banabagatɔw jira minnu bɛ bɛn ɲɔgɔn ma (n = 6). FMO, fluorescence min bɛ bɔ kelen na; MFI, o ye fluorescence fanga cɛmancɛ ye.
Sɛgɛsɛgɛli wɛrɛw y’a jira ko danfaraba wɛrɛw bɛ T-cell fenotipiki cogoya ɲɛnabɔlenba ni ɲɔgɔn cɛ. Hakilila min bɛ baara kɛ (Jaa 1, G ka se I ma) ani hakilijagabɔ kɛcogo (Jaa 1, J ani K) kuruw la, olu ka ca kosɛbɛ ka tɛmɛ asidi kan (CD3 + T selilɛriw hakɛ). O cogo kelen na, ka fenotipi sɛgɛsɛgɛ ni activation markers (CD25 ani CD137) ani depletion markers [programmé cellule death proteine ​​1 (PD1)] jirali ye, o y’a jira ko hali ni nin jamana ninnu ka farikoloɲɛnajɛ kɛcogo tɛ kelen ye (Ja S1, B ka taa E) , Nka danfaraba si ma ye farikoloɲɛnajɛ la tuma bɛɛ naive, effector walima memory subsets cɛ (Ja S1, F ka taa I la). Nin jaabi ninnu tiɲɛna ni masin kalan fɛɛrɛw ye walasa ka selilu fɛnɲɛnamafagalanw di u yɛrɛ ma (21), o min y’a jira ka t’a fɛ ko kolotugudaw caman (CD45 + / CD3- / CD4 + / CD45RO +) bɛ banabagatɔ ka sɔgɔsɔgɔninjɛ la (Ja S2A ). Kɔnɔnatumu suguya minnu dɔnna, olu bɛɛ la, nin selilu miyɛloyidi in ye sukaro tali ni mitokondriyaw ka baara caya jira (Jaa S2, B ka taa G la). Nin jaabi ninnu bɛ danfara barikama jira farikoloɲɛnajɛ la farikolokisɛ suguya caman ni ɲɔgɔn cɛ minnu bɛ sɔrɔ sɔgɔsɔgɔninjɛ ni kuruw la HGSC banabagatɔw la.
Gɛlɛya belebele min bɛ TIL ka farikoloɲɛnajɛ kɛcogo faamuyali la, o ye a ka kan ka T-cell sɛgɛsɛgɛli kɛ minnu saniyalen don, minnu ka ɲi ani minnu hakɛ bɛ se ka kɛ, ka bɔ tumuw la. Kɔsa in na, sɛgɛsɛgɛliw y’a jira ko fɛnw tilatilali ni biɲɛw falenfalencogo minnu sinsinnen bɛ flow cytometry kan, olu bɛ se ka kɛ sababu ye ka fɛn caman Changer cellulaire metabolite profiles (22-24). Walasa ka se sɔrɔ nin gɛlɛya in kan, an ye biɲɛ falencogo ɲuman kɛ walasa ka TIL bɔ hadamadenw ka kɔnɔbara kansɛri la min tigɛra opereli fɛ, ka sɔrɔ ka sɛgɛsɛgɛli kɛ ni LC-MS/MS ye (aw ye sɛbɛn ɲɛ Fɛnw ni fɛɛrɛw lajɛ; Jaa 2A). Walasa ka nin sariyasun in nɔ bɛɛ lajɛlen jateminɛ farikolojɔlifɛnw caman cili la, an ye T-cell minnu bɛ baara kɛ ni dɛmɛbaga kɛnɛmanw ye, olu ka farikolojɔlifɛnw cogoyaw suma ɲɔgɔn na sanfɛ biɲɛw farali taabolo kɔfɛ ni seliluw ye minnu ma faranfasi ni biɲɛ ye nka u tora jikuruw kan. Nin jogoɲini sɛgɛsɛgɛli in y’a jira ko jɛɲɔgɔnya caman bɛ nin cogoya fila ninnu cɛ (r = 0,77), wa, farikolojɔlifɛn 86 kulu ka fɛɛrɛ seginni bɛ se ka segin-ka-bɔ kosɛbɛ (Jaa 2B). O la, nin fɛɛrɛ ninnu bɛ se ka farikolojɔlifɛnw sɛgɛsɛgɛli tigitigi kɛ seliluw la minnu bɛ selilu suguya falenfalen kɛ, o la, u bɛ se ka kɛ sababu ye ka farikolojɔlifɛn kɛrɛnkɛrɛnnenw dɔn HGSC kɔnɔ, o cogo la, u bɛ se ka faamuyali jugu sɔrɔ selilu kɛrɛnkɛrɛnnenw kan Cɛnimusoko farikoloɲɛnajɛ porogaramu.
(A) Ja min bɛ kɛ ni magnɛti biɲɛw falenfalenni ye. Sani u ka sɛgɛsɛgɛli kɛ ni LC-MS/MS ye, seliluw bɛna kɛ ni magɛnni biɲɛw falenni ye siɲɛ saba tugu-tugu ɲɔgɔn na walima u bɛna to jikuruw kan. (B) Nafa suguya bɛ nɔ min bila farikolojɔlifɛnw caya la. Sumanikɛlan saba cɛmancɛ la nafolo suguya kelen-kelen bɛɛ la ± SE. Layini jɛman bɛ jɛɲɔgɔnya jira 1:1. Sumani segin-seginlenw ka jɛɲɔgɔnya (ICC) min jiralen bɛ aksidan taamasiyɛn na. NAD, nikotinamidi adenini dinukiliyɔtidi. (C) Banabagatɔw ka farikolojɔlifɛnw sɛgɛsɛgɛli baarakɛcogo jatebɔsɛbɛn. Asiti walima kurukuruw bɛ lajɛ banabagatɔw la ka u mara sumaya la. Sɛgɛsɛgɛli kelen-kelen bɛɛ yɔrɔ fitinin dɔ sɛgɛsɛgɛra ni flow cytometry ye, ka sɔrɔ sɛgɛsɛgɛli tɔw kɛra nafolotigiya siɲɛ saba ye CD4+, CD8+ ani CD45- selilɛriw la. O selilu tilayɔrɔw sɛgɛsɛgɛra ni LC-MS/MS ye. (D) Funteni karti min bɛ farikolojɔlifɛnw caya hakɛ dantigɛlen jira. Dendrogramme bɛ Ward ka Euclidean yɔrɔjanw kulusi jira misaliw ni ɲɔgɔn cɛ. (E) Principal component analysis (PCA) of sample metabolite map, ka sample kelen-kelen bɛɛ lajɛlen saba jira, samples minnu bɔra banabagatɔ kelen na, olu bɛ ɲɔgɔn cɛ ni layini ye. (F) PCA min bɛ kɛ ni fɛnɲɛnamafagalanw cogoya ye, min bɛ kɛ ni sɛgɛsɛgɛli kɛlen ye banabagatɔ kan (o kɔrɔ ye ko ka baara kɛ ni redundancy partielle ye); misali suguya dan ye a ka kurunba kɔnɔna ye min bɛ wele ko convex. PC1, o ye yɔrɔba 1 ye; PC2, o ye yɔrɔba 2 ye.
O kɔfɛ, an ye nin nafolosɔrɔ fɛɛrɛ in kɛ ka farikolojɔlifɛn 99 sɛgɛsɛgɛ CD4 +, CD8 + ani CD45-cell fractions kɔnɔ HGSC banabagatɔ wɔɔrɔ ka ascite fɔlɔw ni kurukuruw la (Jaa 2C, Jaa S3A ani Tablo S3 ani S4). Nafa hakɛ bɛ kɛ 2% fo 70% ye farikolokisɛ ɲɛnamaw hakɛ fɔlɔ la, wa farikolokisɛw hakɛ bɛ danfaraba don banabagatɔw ni ɲɔgɔn cɛ. Biɲɛw faralen kɔfɛ, nafa tilayɔrɔba min falen don (CD4+, CD8+ walima CD45-) o bɛ tɛmɛ 85% kan farikolokisɛ ɲɛnamaw bɛɛ la minnu bɛ sɛgɛsɛgɛli la, ni u kɛra ɲɔgɔn cɛ. Nin nafolosɔrɔcogo in b’a to an bɛ se ka selilu hakɛw sɛgɛsɛgɛ ka bɔ hadamadenw ka tumuw farikolojɔli la, o min tɛ se ka kɛ ka bɔ fɛnba dɔw la. An kɛlen ka baara kɛ ni nin sariyasen in ye, an y’a ye ko l-kynurenine ni adenosine, nin farikolo tangalan fila minnu taamasiɲɛ ka ɲi, olu kɔrɔtara tumu T selilɛriw walima tumu selilɛriw kɔnɔ (Jaa S3, B ani C). O la, nin jaabi ninnu b’a jira ko an ka selilu faranfasicogo ni mass spectrometry fɛɛrɛ ye kantigiya n’a seko ye walasa ka farikolojɔlifɛnw sɔrɔ minnu nafa ka bon ɲɛnamaya ta fan fɛ banabagatɔw ka farikolo yɔrɔw la.
An ka sɛgɛsɛgɛli fana y’a jira ko farikolokisɛ suguyaw faralen don ɲɔgɔn kan kosɛbɛ banabagatɔw kɔnɔ ani banabagatɔw ni ɲɔgɔn cɛ (Jaa 2D ani ja S4A). Kɛrɛnkɛrɛnnenya la, ni aw ye a suma ni banabagatɔ tɔw ye, banabagatɔ 70 ye farikoloɲɛnajɛ kɛcogo wɛrɛw jira (Ja 2E ani Jaa S4B), o b’a jira ko farikoloɲɛnajɛ suguya caman bɛ se ka kɛ banabagatɔw ni ɲɔgɔn cɛ. A ka kan ka kɔlɔsi ko ni i ye a suma ni banabagatɔ tɔw ye (litiri 1,2 ka se litiri 2 ma; Tablo S1), asidi hakɛ bɛɛ lajɛlen min lajɛra banabagatɔ 70 la (mililitiri 80) o tun ka dɔgɔn. Banabagatɔw ni ɲɔgɔn cɛ ɲɔgɔndan kɔlɔsili kɛli yɔrɔ kolomaw sɛgɛsɛgɛli senfɛ (misali la, ka baara kɛ ni sɛgɛsɛgɛli ye min bɛ kɛ ni fɛn dɔw ye) bɛ fɛn caman jira minnu bɛ ɲɔgɔn falen-falen selilu suguyaw ni ɲɔgɔn cɛ, wa selilu suguyaw ni/walima sigida fitininw bɛ fara ɲɔgɔn kan ka jɛya ka kɛɲɛ ni farikolojɔlifɛnw cogoya ye (Ja 2F ). Fɛnɲɛnɛmafagalan kelenw sɛgɛsɛgɛli ye sinsin kɛ o nɔ ninnu kan, wa a ye danfaraba jira selilu suguyaw ni sigida fitininw cɛ. A ka kan ka kɔlɔsi ko danfara min ka bon kosɛbɛ, o ye MNA ye, o min ka teli ka kɛ CD45- selilɛriw la ani CD4+ ni CD8+ selilɛriw la minnu bɛ don tumu kɔnɔ (Jaa 3A). CD4 + selilɛriw ta fan fɛ, o nɔ in bɛ ye kosɛbɛ, wa a bɛ iko MNA min bɛ CD8 + selilɛriw kɔnɔ, o fana bɛ nɔba bila sigida la. Nka, o nafa tɛ, barisa banabagatɔ wɔɔrɔ la, saba dɔrɔn de bɛ se ka jateminɛ kɛ tumo CD8+ hakɛw la. Ka fara MNA kan, farikolokisɛ suguya wɛrɛw la minnu bɛ sɔgɔsɔgɔninjɛ ni kuruw la, farikolojɔlifɛn wɛrɛ minnu tɛ dɔn kosɛbɛ TIL la, olu fana nafa ka bon cogoya la (Jaa S3 ni S4). O la, nin kunnafoniw bɛ farikolo tangalanw kulu dɔ jira min bɛ layidu ta walasa ka ɲinini wɛrɛw kɛ.
(A) MNA hakɛ bɛnnen don CD4+, CD8+ ani CD45- selilɛriw la minnu bɛ bɔ asidi ni kuru la. Box plot bɛ cɛmancɛ (layini), cɛmancɛ-yɔrɔ (frame hinge) ani kunnafoni-falen-yɔrɔ jira, fo ka se 1,5 ma ka tɛmɛ 1,5 ni ɲɔgɔn cɛ (frame whisker). I n’a fɔ a ɲɛfɔlen don cogo min na banabagatɔ ka minɛnw ni a kɛcogo la, aw bɛ banabagatɔ ka limma hakɛ kɛ walasa ka P hakɛ dɔn (*P<0.05 ani **P<0.01). (B) MNA farikoloɲɛnajɛ ja (60). Fɛnɲɛnamafagalanw: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine, n’o ye S-adenosine-1-homocysteine ​​ye; NA, nikotinamidi, nikotinamidi; MNA, 1-metilnikotinamidi; 2-PY, 1-metil- 2-piridɔni-5-karɔbɔksamidi; 4-PY, 1-metil-4-piridɔni-5-karɔbɔksamidi; NR, nikotinamidi ribose; NMN, nikotinamidi mononukiliyɔtidi. Enzymes (bulu jɛmanw): NNMT, nikotinamidi N-metiltransferase; SIRT, n’o ye sirtuins ye; NAMPT, nikotinamidi fosforibosili transferase; AOX1, aldehidi ɔkisidazi 1; NRK, nikotinamidi ribosidi kinase; NMNAT, nikotinamidi mono Nukilitɔn adenilati transferase; Pnp1, purini nukilisi fosforilasi. (C) t-SNE ka scRNA-seq ka ascites (gris) ani tumor (bilen; n = banabagatɔ 3). (D) NNMT jirali selilu jamana danfaralenw na minnu dɔnna ni scRNA-seq ye. (E) NNMT ni AOX1 jirali SK-OV-3 la, hadamaden denw ka sugunɛ (HEK) 293T, T-cell ani T-cell minnu furakɛra ni MNA ye. A fɔcogo folilen jiralen don ka kɛɲɛ ni SK-OV-3 ye. Ɲɛjirali kɛcogo ni SEM ye, o jiralen don (n = 6 dibaga kɛnɛmanw). Ct hakɛ minnu ka ca ni 35 ye, olu bɛ jate ko u tɛ se ka dɔn (UD). (F) SLC22A1 ni SLC22A2 jirali SK-OV-3, HEK293T, T selilɛriw ani T selilɛriw la minnu furakɛra ni 8mM MNA ye. A fɔcogo folilen jiralen don ka kɛɲɛ ni SK-OV-3 ye. Ɲɛjirali kɛcogo ni SEM ye, o jiralen don (n = 6 dibaga kɛnɛmanw). Ct hakɛ minnu ka ca ni 35 ye, olu bɛ jate ko u tɛ se ka dɔn (UD). (G) Kɔnɔnatumuw ka MNA hakɛ bɛ selilu T kɛnɛmanw na minnu bɛ baara kɛ, lɛri 72 kɔnɔ, u kɛlen kɔ ka MNA kɛ. Ɲɛjirali kɛcogo ni SEM ye, o jiralen don (n = 4 dibaga kɛnɛmanw).
MNA bɛ sɔrɔ ni metil kulu jiginni ye ka bɔ S-adenosyl-1-methionine (SAM) la ka taa nikotinamidi (NA) la nikotinamidi N-methyltransferase fɛ (NNMT; Jaa 3B). NNMT bɛ sɔrɔ ka tɛmɛ hadamadenw ka kansɛri suguya caman kan, wa a bɛ tali kɛ a caya, a doncogo ani a jiginni na (25-27). Walasa ka MNA sɔrɔyɔrɔ faamuya ka ɲɛ T selilɛriw la TME kɔnɔ, an ye scRNA-seq kɛ ka NNMT jiracogo jira selilu suguyaw bɛɛ la HGSC banabagatɔ saba ka sɔgɔsɔgɔninjɛ ni kuruw la (Taabolo S5). Sɛgɛsɛgɛli kɛra farikolokisɛ 6500 ɲɔgɔn kan, o y’a jira ko sɔgɔsɔgɔninjɛ ni tumu sigidaw la, NNMT jirali dan ye fibroblaste ni tumokisɛw hakɛ jateminɛlenw ye (Jaa 3, C ani D). A ka kan ka kɔlɔsi ko NNMT jiracogo jɛlen tɛ jamana si la min bɛ PTPRC (CD45 +) jira (Ja 3D ani ja S5A), o b’a jira ko MNA min sɔrɔla farikolojɔlifɛnw na, o donna T selilɛriw kɔnɔ. Aldehyde oxidase 1 (AOX1) jirali bɛ MNA caman tigɛli kɛ 1-metil-2-piridone-5-carboxamide (2-PYR) walima 1-methyl-4-pyridone-5-carboxamide (4- PYR) ye Jaa 3B) fana dan ye fiɲɛbɔlanw hakɛ ye minnu bɛ COL1A1 jira (Jaa S5A), olu bɛɛ lajɛlen b’a jira ko T-cellw tɛ se ka MNA sɛnɛcogo kɔrɔ kɛ. Nin MNA ni ɲɔgɔn cɛ jamu ninnu jiracogo sɛgɛsɛgɛra ni selilɛri yɛrɛmahɔrɔnyalen kunnafonidilan filanan ye min bɔra asidi la ka bɔ HGSC banabagatɔw la (Jaa S5B; n = 6) (16). Ka fara o kan, quantitative polymerase chain reaction (qPCR) sɛgɛsɛgɛli kɛra T-cell kɛnɛmanw kan minnu furakɛra ni MNA ye, o y’a jira ko ni i ye a suma ni SK-OV-3 kɔnɔbara tumu selilɛriw ye minnu bɛ kɔrɔsi, NNMT walima AOX1 bɛ ɲini ka jira (Jaa 3E). Nin jaabi minnu ma labɛn, olu b’a jira ko MNA bɛ se ka bɔ fiɲɛbanakisɛw walima tumuw la ka don T-cell kɛrɛfɛtaw la TME kɔnɔ.
Hali ni kandidaw ye katiyɔn 1 fo 3 (OCT1, OCT2 ani OCT3) denbaya ye minnu kodɔnna soluble carrier 22 (SLC22) denbaya fɛ (SLC22A1, SLC22A2 ani SLC22A3), MNA tabaga minnu bɛ se ka kɛ, olu ma ɲɛfɔ hali bi (28) ARNm min bɔra T-cell kɛnɛmanw na, o ka QPCR ye SLC22A1 jiracogo dɔgɔya jira nka SLC22A2 hakɛ min tɛ se ka dɔn, o y’a jira ko a fɔra ka tɛmɛ sɛbɛnw kɔnɔ (Ja 3F) (29). O ni SK-OV-3 ovari tumor cell line ta tɛ kelen ye, o ye transporteur fila bɛɛ hakɛ caman jira (Jaa 3F).
Walasa ka se ka T-cellw sɛgɛsɛgɛ ni se bɛ u ye ka dunan MNA minɛ, T-cell kɛnɛmanw ladonna sanga 72 kɔnɔ MNA hakɛ danfaralenw na. Ni MNA kɔkanna tɛ yen, MNA selilɛri kɔnɔfɛnw tɛ se ka dɔn (Jaa 3G). Nka, T-cell activés minnu furakɛra ni MNA kɛnɛma ye, olu ye dɔ fara MNA hakɛ kan seliluw kɔnɔ ka kɛɲɛ ni a tata hakɛ ye, fo ka se MNA 6 mM ma (Jaa 3G). Nin jaabi in b’a jira ko hali ni transporteur (taamakɛla) jirali hakɛ ka dɔgɔ ani anzimu kunba min bɛ MNA sɛnɛcogo ɲɛnabɔ selilɛri kɔnɔ, o tɛ yen, TIL bɛ se ka MNA ta hali bi.
Banabagatɔw ka T-cell kɔnɔ farikolojɔlifɛnw ka spektrum ani MNA minɛni kɔlɔsiliw in vitro, o bɛ dɔ fara a kan ko kansɛri ni fibroblaste (CAF) bɛ MNA bɔ ani tumu selilɛriw bɛ se ka TIL ka fɛnɲɛnamafagalan ni a baaracogo ɲɛnabɔ. Walasa ka MNA nɔ dɔn T-cellw kan, T-cell dibagaw kɛnɛmanw kɛra baara ye in vitro MNA bɛ yen walima ni u tɛ yen, ani u caya ni sitokini sɔrɔli jateminɛna. Tile 7 MNA faralen kɔfɛ a tata hakɛ cayalenba la, jamanadenw hakɛ cayalenba siɲɛ fila dɔgɔyara dɔɔni dɔɔni, ka sɔrɔ fanga tora a tata hakɛ bɛɛ la (Jaa 4A). Ka fara o kan, MNA kɛnɛma furakɛli kɛra sababu ye ka dɔ fara CD4 + ni CD8 + T selilɛriw hakɛ kan minnu bɛ tumor necrosis factor-α (TNFα; Jaa 4B) jira. O ni IFN-γ bɔli selilɛri kɔnɔ, o dɔgɔyara kosɛbɛ CD4 + T selilɛriw la, nka a ma dɔgɔya CD8 + T selilɛriw la, wa fɛn caman ma Changé interleukin 2 la (IL-2; Jaa 4, C ani D). O la, enzyme-linked immunosorbent assay (ELISA) min bɛ kɛ ni supernatants ye ka bɔ nin T-cell cultures ninnu na minnu furakɛra ni MNA ye, o ye dɔ fara TNFα kan kosɛbɛ, IFN-γ dɔgɔyara, ani IL-2 ma Changé (Jaa 4, E ka taa G la). . IFN-γ dɔgɔyali b’a jira ko MNA bɛ se ka jɔyɔrɔba ta T-cellw ka tumu kɛlɛli baara balili la. Walasa ka MNA nɔ min bɛ T-cell-mediated cytotoxicity (T-cell-mediated cytotoxicity) la, o ladege, chimeric antigen receptor T (FRα-CAR-T) selilu minnu bɛ folate minɛbaga α ni CAR-T (GFP) laɲini minnu bɛ ɲɛnabɔ ni green fluorescent protein (GFP) -CAR-T) selilɛriw ye, olu bɛ dilan ni donna periferik joli mononucléaire cellules (PBMC) kɛnɛmanw ye. CAR-T seliluw ladonna sanga 24 kɔnɔ MNA ɲɛkɔrɔ, o kɔfɛ, u ni hadamaden SK-OV-3 kɔnɔbara tumu selilɛriw kɛra ɲɔgɔn fɛ minnu bɛ folate minɛbaga α jira ni effecteur ni target hakɛ ye 10:1 ye. MNA furakɛli kɛra sababu ye ka FRα-CAR-T selilɛriw fagali baara dɔgɔya kosɛbɛ, o ni FRα-CAR-T selilɛriw furakɛra ni adenosini ye (Jaa 4H).
(A) Total viable cell count ani population doubling (PD) directement ka bɔ culture la tile 7. Bar graph bɛ dɛmɛbaga kɛnɛman wɔɔrɔ ka cɛmancɛ + SEM jira. A bɛ kunnafoniw jira minnu bɔra n = 3 la dɔgɔmannin yɛrɛmahɔrɔnya kɔlɔsiliw la. (B ka taa D) CD3/CD28 ni IL-2 kɛra ka T-cellw baara u ka MNA hakɛ kelen-kelen bɛɛ la tile 7 kɔnɔ. Sani sɛgɛsɛgɛli ka kɛ, seliluw lawulila ni PMA/ionomycin ye ni GolgiStop ye sanga 4 kɔnɔ. TNFα (B) jirali T-cellw la. Misali ja (numan fɛ) ani tabali kunnafoniw (niman fɛ) TNFα jiracogo kan farikolokisɛ ɲɛnamaw la. IFN-γ (C) ni IL-2 (D) jirali T selilɛriw la. Sitokiniw jiracogo suman ni flow cytometry ye. Bar graph bɛ hakɛ danmadɔ jira (n = 6 dɛmɛbaga kɛnɛmanw) + SEM. Aw bɛ baara kɛ ni ɲɔgɔndan sɛgɛsɛgɛli ye sira kelen fɛ ani sumanikɛlan segin-seginlenw (*P<0.05 ani **P<0.01) walasa ka P hakɛ dɔn. A bɛ kunnafoniw jira minnu bɔra n = 3 yɛrɛmahɔrɔnyalenw na. (E ka taa G) CD3/CD28 ni IL-2 kɛra ka T-cellw baara u ka MNA hakɛ kelen-kelen bɛɛ la tile 7 kɔnɔ. Aw bɛ furaji lajɛ ka kɔn PMA/ionomycine lawulili ɲɛ ani sanga 4 kɔfɛ. TNFα (E), IFN-γ (F) ani IL-2 (G) hakɛw jateminɛna ni ELISA ye. Bar graph bɛ hakɛ danmadɔ jira (n = 5 kɛnɛya dibaga) + SEM. P hakɛ min latigɛra ni sira kelen fɛ sɛgɛsɛgɛli ye min bɛ kɛ ni danfara ye ani sumani segin-seginw (*P<0.05). Layini nɔrɔlen bɛ detekti dɔnni dan jira. (H) Kɔnɔnatumuw sɛgɛsɛgɛli. FRα-CAR-T walima GFP-CAR-T selilɛriw ladilanna ni adenosini (250μM) walima MNA (10 mM) ye sanga 24 kɔnɔ, walima u ma furakɛ (Ctrl). SK-OV-3 banakisɛw fagali kɛmɛsarada la, o jateminɛna. P hakɛ min bɛ sɔrɔ Welch t sɛgɛsɛgɛli fɛ (*P<0,5 ani **P<0,01).
Walasa ka fɛɛrɛ faamuyali sɔrɔ TNFα jiracogo ɲɛnabɔli la min bɛ tali kɛ MNA la, fɛn minnu bɛ TNFα mRNA la, olu jateminɛna T-cell minnu furakɛra ni MNA ye, olu jateminɛna (Jaa 5A). T-cell kɛnɛmanw minnu furakɛra ni MNA ye, olu ye dɔ fara TNFα sɛbɛnni hakɛ kan siɲɛ fila, o b’a jira ko MNA bɛ tali kɛ TNFα sɛbɛnni ɲɛnabɔli la. Walasa ka nin ɲɛnabɔli fɛɛrɛ in sɛgɛsɛgɛ min bɛ se ka kɛ, sɛbɛnnikɛlan dɔnnen fila minnu bɛ TNFα ɲɛnabɔ, n’olu ye T cellule nucléaire activé (NFAT) ani poroteyini kɛrɛnkɛrɛnnen 1 (Sp1) ye, olu jateminɛna ka kɛɲɛ ni MNA sirili ye TNFα promotɛri kɛrɛfɛ ( 30 ). TNFα promotɛri kɔnɔ, NFAT siriyɔrɔ 6 dɔnna ani Sp1 siriyɔrɔ 2, minnu bɛ ɲɔgɔn dafa yɔrɔ kelen na [-55 basi fila (bp) ka bɔ 5’cap la] (30). Chromatin immunoprecipitation (ChIP) y’a jira ko ni a furakɛra ni MNA ye, Sp1 sirili TNFα promotɛri la, o bɛ bonya siɲɛ saba. NFAT donli fana ye dɔ fara a nafa kan, wa a gɛrɛla a la (Jaa 5B). Nin kunnafoniw b’a jira ko MNA bɛ TNFα jiracogo ɲɛnabɔ Sp1 sɛbɛnni fɛ, ani ka dɔgɔya ka tɛmɛ NFAT jirali kan.
(A) Ni an ye a suma ni T selilɛriw ye minnu falen don MNA tɛ minnu na, TNFα jiracogo caman caman cili T selilɛriw la minnu furakɛra ni MNA ye. Ɲɛjirali kɛcogo ni SEM ye, o jiralen don (n = 5 dibaga kɛnɛmanw). A bɛ kunnafoniw jira minnu bɔra n = 3 yɛrɛmahɔrɔnyalenw na. (B) T-cell minnu furakɛra ni 8 mM MNA ye walima ni u tɛ minnu na NFAT ni Sp1 kɔfɛ, olu ka TNFα promotɛri farala ɲɔgɔn kan ni (Ctrl) ni PMA/ionomycine lawulili ye sanga 4 kɔnɔ. Immunoglobuline G (IgG) ni H3 kɛra banakisɛfagalanw ye minnu tɛ se ka furakɛ ani minnu bɛ se ka kɛ sababu ye ka farikolo tangacogo ɲɛnabɔ. ChIP hakɛ jateminɛni y’a jira ko Sp1 ni NFAT sirili TNFα promotɛri la MNA furakɛli kɛlenw na, o cayara siɲɛ caman ni a ni kɔrɔsili ta kɛra ɲɔgɔn fɛ. A bɛ kunnafoniw jira minnu bɔra n = 3 la dɔgɔmannin yɛrɛmahɔrɔnya kɔlɔsiliw la. P hakɛ min bɛ sɔrɔ t-test caman fɛ (*** P <0,01). (C) Ni an ye a suma ni HGSC ka ascites ye, T cellules (minnu tɛ sitotokisi ye) ye TNF jirali caya jira tumu kɔnɔ. Kulɛriw bɛ banabagatɔ suguya caman jira. Kɔnɔnatumu minnu jirala, olu ta kɛra k’u kɛ 300 ye k’u jija walasa ka dan sigi u bɔli la kojugu (** Padj = 0,0076). (D) MNA modɛli sugandilen kɔnɔbara kansɛri kama. MNA bɛ sɔrɔ tumuw la ani fibroblaste (fibroblaste) TME kɔnɔ, wa a bɛ ta T-cell fɛ. MNA bɛ dɔ fara Sp1 sirili kan TNFα promotɛri la, o bɛ na ni TNFα sɛbɛnni caya ye ani TNFα sitokini bɔli. MNA fana bɛ kɛ sababu ye ka IFN-γ dɔgɔya. T-cell baara balili bɛ na ni mɔgɔfaga seko dɔgɔyali ye ani ka tumuw falenni teliya.
Ka kɛɲɛ ni kunnafoniw ye, TNFα bɛ ni ɲɛfɛla ni kɔ la, tumu ni tumu kɛlɛli nɔ bɛ a la, nka a jɔyɔrɔ dɔnna kosɛbɛ kɔnɔbara kansɛri bonya ni a jiginni na (31-33). Ka kɛɲɛ ni kunnafoniw ye, TNFα hakɛ bɛ sɔrɔ sɔgɔsɔgɔninjɛ ni tumuw la banabagatɔw la minnu bɛ kɔnɔbara kansɛri la, o ka ca ni farikolo yɔrɔ ɲumanw ta ye (34-36). Fɛɛrɛ siratigɛ la, TNFα bɛ se ka joli kuru finmanw baaracogo, u baaracogo ani u cayacogo ɲɛnabɔ, ani ka kansɛribanakisɛw cogoya Changer (37, 38). Ka bɛn ni nin sɔrɔlen ninnu ye, diferential gene expression analysis y’a jira ko TNF tun bɛ wuli kosɛbɛ T selilɛriw la tumuw kɔnɔ ni i ye a suma ni ascite ye (Jaa 5C). TNF jirali cayalenba tun bɛ ye T-cell jamanadenw dɔrɔn de la minnu ka fɛnɲɛnamafagalan tɛ sitotokisi ye (Jaa S5A). Kuma surun na, nin kunnafoniw bɛ hakilina in sinsin ko MNA bɛ ni farikolo tangalan ni tumuw sabatili nɔ fila ye HGSC la.
Fluorescent labeling min sinsinnen bɛ flow cytometry kan, o kɛra fɛɛrɛba ye min bɛ kɛ ka TIL farikoloɲɛnajɛ kalan. Nin ɲinini ninnu y’a jira ko ni i ye a suma ni joli laminiw ye walima ni T-cell minnu bɛ bɔ farikolo yɔrɔ filananw na, sosow ni hadamadenw ka TIL ka teli ka sukaro ta (4, 39) ani ka mitokondriyaw ka baara bɔnɛ dɔɔni dɔɔni (19, 40). Hali n’an ye o jaabi kelenw kɔlɔsi nin ɲinini in na, yiriwali kunba ye ka tumuw ni TIL farikolojɔli suma ɲɔgɔn na ka bɔ tumokisɛ kelen tigɛlen kelen na. Ka bɛn ni nin kunnafoni tɛmɛnen dɔw ye, tumu (CD45-EpCAM +) banakisɛ minnu bɛ bɔ asidi ni tumuw la, olu ka sukaro tali ka ca ka tɛmɛ CD8 + ni CD4 + T selilɛriw kan, o b’a jira ko tumuw ka sukaro caman tali bɛ se ka suma ni T selilɛriw ye. T-cell ka ɲɔgɔndan hakilina. TME ye. Nka, tumuw ka mitokondriyaw ka baara ka ca ni CD8 + T selilɛriw ta ye, nka mitokondriyaw ka baara ni CD4 + T selilɛriw ta bɛ tali kɛ ɲɔgɔn na. Nin jaabi ninnu bɛ dɔ fara barokun min bɛ ka bɔ kɛnɛ kan, n’o ye ko oksidan farikoloɲɛnajɛ nafa ka bon tumuw la (41, 42). U b’a jira fana ko CD8 + T selilɛriw bɛ se ka kɛ sababu ye ka oksidan baarakɛbaliya sɔrɔ ka tɛmɛ CD4 + T selilɛriw kan, walima ko CD4 + T selilɛriw bɛ se ka baara kɛ ni karɔbɔli sɔrɔyɔrɔw ye minnu tɛ sukaro ye walasa ka mitokondriyaw ka baara sabati (43, 44). A ka kan ka fɔ ko an ma danfara si kɔlɔsi sukaro tali walima mitokondriyaw ka baara la CD4 + T effecteurs, T effecteur memory ani T central memory cellules cɛ ascites la. O cogo kelen na, CD8 + T selilɛriw ka danfara cogoya tɛ tumuw la, o ni sukaro tali yeli ɲɔgɔn tɛ, o bɛ danfaraba jira T selilɛriw cɛ minnu falen don in vitro ni hadamaden ka TIL in vivo (22). Nin kɔlɔsili ninnu fana sinsinnen don ni selilu hakɛ tilatilali ye min tɛ mɔgɔ bɔ mɔgɔ la, o min y’a jira ka t’a fɛ ko CD45 + / CD3- / CD4 + / CD45RO + selilu minnu ka sukaro tali ni mitokondriyaw ka baara ka ca ni tumuw ye, olu ka ca nka Metabolic active cell population bɛ u la. Nin jamana in bɛ se ka kɛ miyɛloyi suppresseur cellules walima plasmacytoïde dendritic cellules ka jamanaden fitininw ye minnu jirala scRNA-seq sɛgɛsɛgɛli la. Hali ni nin fila bɛɛ fɔra hadamadenw ka kɔnɔbara basigilenw na [45], u mago bɛ hali bi Baara wɛrɛ ye ka nin miyɛloyi jamanaden fitinin in ɲɛfɔ.
Hali ni fɛɛrɛw minnu sinsinnen bɛ flow cytometry kan, olu bɛ se ka danfara caman ɲɛfɔ sukaro ni oksidan sɛnɛcogo la selilu suguyaw ni ɲɔgɔn cɛ, farikolojɔlifɛn tigitigi minnu bɛ bɔ sukaro walima karɔbɔni sɔrɔyɔrɔ wɛrɛw fɛ mitokondriyaw farikoloɲɛnajɛ kama TME kɔnɔ, olu ma dɔn fɔlɔ. Ka farikolojɔlifɛnw sɔrɔli walima u sɔrɔbaliya di TIL kulu fitinin dɔ ma, o bɛ a ɲini ka selilu jamana saniya ka bɔ farikolo yɔrɔ tigɛlen na. O la, an ka selilɛriw falenfalen fɛɛrɛ min farala ɲɔgɔn kan ni mass spectrometry ye, o bɛ se ka hakilinaw di farikolojɔlifɛnw kan minnu falen don danfara la T selilɛriw ni tumu selilɛriw jamanaw na banabagatɔw ka misaliw la minnu bɛ bɛn ɲɔgɔn ma. Hali ni nafa bɛ nin fɛɛrɛ in na ka tɛmɛ selilɛriw tilatilali kan minnu bɛ baara kɛ ni fluorescence ye, farikolojɔlifɛnw gafemarayɔrɔ dɔw bɛ se ka nɔ bila a la ka da a sabatili danma kan ani/walima u jiginni teliya la (22). O bɛɛ n’a ta, an ka fɛɛrɛ sera ka farikolo tangalan fila dɔn minnu dɔnna, n’olu ye adenosini ni kynurenine ye, bawo u bɛ danfaraba don misali suguyaw ni ɲɔgɔn cɛ.
An ka fɛnɲɛnamafagalanw sɛgɛsɛgɛli min kɛra tumuw ni TIL suguya fitininw kan, o bɛ hakilina caman di farikolojɔlifɛnw jɔyɔrɔ kan kɔnɔbara TME la. Fɔlɔ, an kɛlen ka baara kɛ ni flow cytometry ye, an y’a ye ko danfara si tɛ mitokondriyaw ka baara la tumuw ni CD4 + T selilɛriw cɛ. Nka. Filanan, MNA ye farikolojɔlan ye min danfara ka bon CD45-cellw ni T-cellw cɛ asiti la, a tɛ kɛ tumuw ye. O la, tilayɔrɔw ni tumuw sigiyɔrɔ bɛ se ka nɔ wɛrɛw bila TIL farikoloɲɛnajɛ la, o min bɛ danfara min bɛ se ka kɛ sigida fitinin dɔ kɔnɔ, o jira. Sabanan, MNA dilanni anzimu NNMT jirali bɛ dan CAF dɔrɔn de ma, o min ye tumuw ye ka dɔgɔya, nka MNA hakɛ minnu bɛ se ka dɔn, olu bɛ ye tumuw la minnu bɛ sɔrɔ tumuw la. NNMT jirali kojugu kɔnɔbara CAF la, o bɛ kansɛri sabatili nɔ dɔn, a dɔ ye CAF farikoloɲɛnajɛ yiriwali ye, tumuw doncogo ani a jiginni (27). Hali ni TIL hakɛ bɛɛ lajɛlen ye danma ye, NNMT jirali CAF la, o ni Cancer Genome Atlas (TCGA) mesenchymal subtype bɛ tali kɛ ɲɔgɔn na kosɛbɛ, o min ni prognosis jugu bɛ tali kɛ ɲɔgɔn na (27, 46, 47). A laban na, anzimu AOX1 min bɛ MNA tiɲɛni ɲɛnabɔ, o fana bɛ dan CAF jamanadenw ma, o b’a jira ko T-cellw tɛ se ka MNA sɛnɛ. Nin jaabi ninnu bɛ hakilina in sinsin ko hali ni baara wɛrɛw ka kan ka kɛ walasa ka nin sɔrɔ in sɛgɛsɛgɛ, MNA hakɛ caman sɔrɔli T-cell kɔnɔ, o bɛ se ka kɛ sababu ye ka CAF microenvironnement min bɛ farikolo tangalan bali, o bɛ yen.
Ni an y’a jateminɛ ko MNA tabagaw ka jirali hakɛ dɔgɔyara ani farikolojɔlifɛn jɔnjɔn minnu bɛ MNA sɛnɛcogo la, olu hakɛ min tɛ se ka dɔn, MNA sɔrɔli T-cell kɔnɔ, o ye ko ye min ma deli ka kɛ. NNMT ni AOX1 si ma se ka sɔrɔ scRNA-seq sɛgɛsɛgɛli fɛ ani qPCR laɲinilen fɛ kulu yɛrɛmahɔrɔnyalen fila la. Nin jaabi ninnu b’a jira ko MNA tɛ dilan T-cell fɛ, nka a bɛ minɛ ka bɔ TME lamini na. Kɔrɔbɔli minnu kɛra in vitro kɔnɔ, olu b’a jira ko T-cellw ka teli ka MNA kɔkanna dalajɛ.
An ka in vitro sɛgɛsɛgɛliw y’a jira ko MNA kɔkanna bɛ TNFα jirali bila T-cellw kɔnɔ ani ka Sp1 sirili sabati TNFα promotɛri la. Hali ni TNFα bɛ tumu ni tumu kɛlɛli baara fila bɛɛ kɛ, kɔnɔbara kansɛri la, TNFα bɛ se ka kɔnɔbara kansɛri bonya (31-33). TNFα neutralisation (neutralisation) kɛli ovarian tumor cellule culture la walima TNFα taamasiɲɛ bɔli sosow ka modeliw la, o bɛ se ka TNFα-mediated inflammatory cytokine production ɲɛ ka taa a fɛ ani ka tumor growth bali (32, 35). O la, o cogo la, MNA min bɛ bɔ TME la, o bɛ se ka kɛ farikolojɔlifɛn ye min bɛ banakisɛfagalanw dɛmɛ, ka tɛmɛ fɛɛrɛ dɔ fɛ min bɛ tali kɛ TNFα la, otokirini (autocrine loop) fɛ, o bɛ kɛ sababu ye ka kɔnɔbara kansɛri sɔrɔli ni a jɛnsɛnni sabati (31). Ka da o seko kan, TNFα balili bɛ ka sɛgɛsɛgɛ i n’a fɔ furakɛli fɛɛrɛ min bɛ se ka kɛ kɔnɔbara kansɛri furakɛli la (37, 48, 49). Ka fara o kan, MNA bɛ CAR-T selilɛriw ka sitotokisisi tiɲɛ ka ɲɛsin kɔnɔbara tumu selilɛriw ma, o bɛ dalilu wɛrɛw di farikolo tangacogo kan MNA fɛ. Nin jaabi ninnu bɛɛ lajɛlen bɛ misali dɔ jira, min kɔnɔ, tumuw ni CAF selilɛriw bɛ MNA bɔ ka don TME kɔkanna na. (i) TNF ka kɔnɔbara kansɛri bonya lawulili fɛ ani (ii) MNA fɛ T-cell sitotokisi baara balili fɛ, o bɛ se ka kɛ sababu ye ka tumu fila nɔ bila (Jaa 5D).
Kuncɛli la, ni selilɛriw falenni teliya la, selilɛri kelenw sigiyɔrɔmali ani farikoloɲɛnajɛ kɛcogo faralen ɲɔgɔn kan, nin ɲinini in ye danfaraba jira farikolo tangacogo la tumuw ni asite selilɛriw cɛ HGSC banabagatɔw la. Nin sɛgɛsɛgɛli bɛɛjɛfanga in y’a jira ko danfara bɛ sukaro tali ni mitokondriyaw ka baara la T-cellw ni ɲɔgɔn cɛ, wa a y’a jira ko MNA ye farikolo tangalan ye min tɛ selilu yɛrɛmahɔrɔnyalen ye. Nin kunnafoniw bɛ nɔ bila TME bɛ nɔ bila cogo min na T selilɛriw ka farikoloɲɛnajɛ la hadamadenw ka kansɛriw la. Hali ni balodɛsɛ sɔrɔli tilennen fɔra T-cell ni kansɛribanakisɛw cɛ, farikolojɔlifɛnw fana bɛ se ka kɛ i n’a fɔ ɲɛnabɔbaga tilennenw walasa ka tumuw ka ɲɛtaa sabati ani ka se ka farikolo tangacogo kɔnɔnafiliw bali. Nin farikolojɔlifɛnw jɔyɔrɔ baarakɛcogo ɲɛfɔli wɛrɛ bɛ se ka fɛɛrɛ wɛrɛw da wuli walasa ka tumuw kɛlɛli farikolo tangacogo ɲɛ.
Banabagatɔw ka sɛgɛsɛgɛliw ni kɛnɛyaso kunnafoniw sɔrɔla BC kansɛri tumuw marayɔrɔ fɛ, min sɛgɛsɛgɛra Kanada farikolo yɔrɔw marayɔrɔ fɛ. Ka kɛɲɛ ni sariya ye min sɔnna BC kansɛri ɲinini jogoɲumanya jɛkulu fɛ ani British Columbia Iniwɛrisite fɛ (H07-00463), banabagatɔw bɛɛ ka sɛgɛsɛgɛliw ni u ka kɛnɛyaso kunnafoniw ye sɔnni sɛbɛn sɔrɔ min bɛ kunnafoni di walima u ye u ka sɔnni bila cogo la. O sɛgɛsɛgɛliw bɛ mara BioBank (BRC-00290) kɔnɔ min sɛgɛsɛgɛlen don. Banabagatɔw ka jogo ɲɛfɔlenw jiralen don jatebɔsen S1 ni S5 kɔnɔ. Walasa ka a tanga kiriyɔmu ma, sɔgɔsɔgɔninjɛ bɛ kɛ ka banabagatɔ ka kurukuruw toli ni masin ye, ka sɔrɔ k’a gɛrɛntɛ ka tɛmɛ filɛri dɔ fɛ min janya ye mikron 100 ye walasa ka selilu kelen suspension sɔrɔ. Banabagatɔ ka sɔgɔsɔgɔninjɛ kɛra santirifugare la ni 1500 ye miniti kɔnɔ miniti 10 kɔnɔ 4°C la walasa ka farikolokisɛw kɛ bɔgɔ ye ani ka a sanfɛji bɔ. Bugun minnu sɔrɔla tumu ni sɔgɔsɔgɔninjɛ fɛ, olu maralen don ni sumaya ye hadamaden ka AB serum 50% kɔnɔ min ma baara kɛ ni funteni ye (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) ani 10% dimetil sulfoxyde kɔnɔ. O selilu kelen suspensions maralen ninnu tun bɛ funfun ka kɛ metaboliki ni metabolite determination ye minnu ɲɛfɔlen don jukɔrɔ.
Fɛnɲɛnɛma dafalen bɛ kɛ ni 0,22 μm filɛri ye 50:50 faralen RPMI 1640: AimV kan. RPMI 1640 + 2,05 mM l-glutamine (Thermo Fisher Scientific) min farala hadamaden ka AB serum 10% kan min ma baara kɛ ni funteni ye (Sigma-Aldrich), Hepes 12,5 mM (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Penisilini Streptomycine (PenStrep) jilama (Thermo Fisher Scientific) ani 50 μMB-mercaptoethanol. AimV (Invitrogen) bɛ fara Hepes milimɛtɛrɛ 20 kan (Thermo Fisher Scientific) ani l-glutamine milimɛtɛrɛ 2 (Thermo Fisher Scientific). Flow cytometer staining buffer tun ye 0,22μm filtered phosphate buffered saline (PBS; Invitrogen) ye min farala 3% kan ni 3% ye min tɛ baara kɛ ni AB hadamadenw ka serum ye (Sigma). Kɔnɔnatumuw falenfalen tampon bɛ kɛ ni PBS filɛri 0,22μm ye, ka fara a kan ni hadamaden AB serum 0,5% ye min ma baara kɛ ni funteni ye (Sigma-Aldrich).
37°C dafalen na, seliluw nɔgɔlen don ni 10 nM MT DR ani 100 μM 2-NBDG ye miniti 30 kɔnɔ. O kɔfɛ, seliluw nɔgɔlen don ni ɲɛnamaya kalali eF506 ye 4°C la miniti 15 kɔnɔ. Aw bɛ seliluw wuli kokura FC Block (eBioscience) ani Brilliant Stain Buffer (BD Biosciences) kɔnɔ, ka u dilan flow cytometer staining buffer kɔnɔ (ka kɛɲɛ ni a dilabaga ka laadilikanw ye), ka u bila miniti 10 kɔnɔ so funteni na. Aw bɛ farikolokisɛw nɔgɔ ni banakisɛfagalanw kulu ye (Taabolo S2) flow cytometry staining buffer kɔnɔ 4°C la miniti 20 kɔnɔ. Aw bɛ seliluw lamaga kokura flow cytometry staining buffer kɔnɔ (Cytek Aurora; 3L-16V-14B-8R configuration) sani aw ka u sɛgɛsɛgɛ. Baara kɛ ni SpectroFlo ni FlowJo V10 ye walasa ka selilu jate kunnafoniw sɛgɛsɛgɛ, ani ka baara kɛ ni GraphPad Prism 8 ye walasa ka kunnafoniw dilan. 2-NBDG ni MT DR ka fluorescence intensité median (MFI) kɛra log-normalized ye, o kɔfɛ, t test fila-fila kɛra ka jateminɛ kɛ walasa ka banabagatɔw jate minnu bɛ bɛn ɲɔgɔn ma. Jamanaden minnu ka ko kɛlenw ma se 40 ma, olu bɛɛ bɔ sɛgɛsɛgɛli la; aw bɛ MFI hakɛ 1 don nafa jugu o juguman na sani aw ka jatebɔ sɛgɛsɛgɛli ni kunnafonidilanw jirali kɛ.
Walasa ka dɔ fara sanfɛla taabolo panɛli bololabaarakɛcogo kan, an ye sɛbɛnni dafalen kɛ ni cogoya dantigɛli jiri ye (FAUST) (21) walasa ka seliw di jamanadenw ma u yɛrɛma, seli salenw bɔlen kɔ FlowJo kɔnɔ. An bɛ bɔli ɲɛnabɔ ni bolo ye walasa ka jamanadenw fara ɲɔgɔn kan minnu bɛ i n’a fɔ u tilalen don cogo jugu la (ka PD1+ ni PD1-tumor selilɛriw fara ɲɔgɔn kan) ani jamanaden maralenw. Sɛgɛsɛgɛli kelen-kelen bɛɛ kɔnɔ, selilu 2% ni kɔ bɛ sɔrɔ a cɛmancɛ la, jamanaden 11 bɛɛ lajɛlen na.
Ficoll gradient density centrifugation kɛra ka PBMC faranfasi ka bɔ joli segin furaw la (STEMCELL Technologies). CD8 + T selilɛriw bɔra PBMC la ni CD8 MicroBeads (Miltenyi) ye, ka u bonya ka kɛ fɛn dafalen ye ni TransAct (Miltenyi) ye dɔgɔkun 2 kɔnɔ ka kɛɲɛ ni a dilabaga ka laadilikanw ye. Aw ye seliluw bila ka jɔ tile 5 kɔnɔ, IL-7 bɛ min na (10 ng/ml; PeproTech), o kɔfɛ, u seginna ka u lawuli ni TransAct ye. Tile 7nan na, ka kɛɲɛ ni a dilannikɛla ka laadilikanw ye, hadamadenw ka CD45 MicroBeads (Miltenyi) kɛra ka farikolokisɛw nafa ka kɛɲɛ ni ɲɔgɔn ye siɲɛ saba tugu-tugu ɲɔgɔn na. Bugunw tila-tilalen don ka kɛ flow cytometry sɛgɛsɛgɛli ye (i n’a fɔ a ɲɛfɔlen don cogo min na sanfɛ), ani selilu miliyɔn kelen tilalen don siɲɛ saba LC-MS/MS sɛgɛsɛgɛli kama. O misaliw kɛra ni LC-MS/MS ye i n’a fɔ a ɲɛfɔlen don cogo min na jukɔrɔ. An ye metabolite hakɛ tununnen jateminɛ ni ion hakɛ ye 1000 ye. Sample kelen-kelen bɛɛ bɛ normalize ni total ion number (TIC) ye, ka convertir logaritme la ani ka normalize automatiquement MetaboAnalystR kɔnɔ ka sɔrɔ ka analyse kɛ.
Banabagatɔ kelen-kelen bɛɛ ka selilɛri kelen sɔgɔlen tun bɛ funu ka sɔrɔ ka a sama ka tɛmɛ 40 μm filɛri fɛ ka kɛ fɛnɲɛnamafagalan dafalen ye (i n’a fɔ a ɲɛfɔlen don cogo min sanfɛ). Ka kɛɲɛ ni a dilannikɛla ka sariya ye, sugandili ɲuman kɛcogo saba tugu-tugulen ni magɛnni biɲɛw farali ye ni MicroBeads (Miltenyi) ye walasa ka CD8+, CD4+ ani CD45- seliluw (jikuruw kan) sɛgɛsɛgɛli kɛlenw nafa bonya. Kuma surun na, seliluw bɛ segin ka wuli selilɛriw falenfalen tampon kɔnɔ (i n’a fɔ a ɲɛfɔlen don cogo min sanfɛ) ka jate. Bugunw tun bɛ tobi ni hadamadenw ka CD8 biɲɛw ye, hadamadenw ka CD4 biɲɛw walima hadamadenw ka CD45 biɲɛw (Miltenyi) 4°C la miniti 15 kɔnɔ, ka sɔrɔ ka u ko ni selilɛriw falenfalen tampon ye. O misali bɛ tɛmɛ LS koloma fɛ (Miltenyi), ka tilayɔrɔ ɲumanw ni tilayɔrɔba juguw lajɛ. Walasa ka dɔ bɔ a kuntaala la ani ka selilɛriw ka kɛnɛya taabolo caya, o kɔfɛ, CD8-yɔrɔ bɛ kɛ ka CD4+ nafabɔli siɲɛ filanan kɛ, ani CD4-yɔrɔ bɛ kɛ ka CD45 nafa sɔrɔli kɔfɛ. Aw bɛ furaji to jisuma kan a farali bɛɛ la.
Walasa ka fɛnw labɛn farikolojɔlifɛnw sɛgɛsɛgɛli kama, u ko siɲɛ kelen ni kɔgɔji nɛnɛman ye, ka sɔrɔ ka mɛtanɔli 80% mililitiri 1 fara sɛgɛsɛgɛli kelen-kelen bɛɛ kan, ka sɔrɔ ka u kɛ vortex ye ka u kɛ snap frozen ye azote jilama kɔnɔ. O sɛgɛsɛgɛli kɛlenw kɛra ni fɛnɲɛnamafagalan saba ye, ka u kɛ santirifuge la ni 14.000 ye miniti kɔnɔ miniti 15 kɔnɔ 4°C la. Fɛnɲɛnamafagalanw bɛ ji min na, o bɛ wuli fo ka a ja. Aw bɛ segin ka farikolojɔlifɛnw kɛ asidi formiki 0,03% 50 μl la, ka u wuli ka u ɲagami, ka sɔrɔ ka u kɛ santirifugu la walasa ka nɔgɔw bɔ.
Aw bɛ farikolojɔlifɛnw bɔ i n’a fɔ a ɲɛfɔlen don cogo min sanfɛ. Aw bɛ ji min bɛ sanfɛ, aw bɛ o bila buteli kɔnɔ min bɛ baara kɛ kosɛbɛ, min bɛ wele ko chromatography liquide, walasa ka metabolomiki ɲinini kɛ. Aw bɛ furakɛli kɛcogo kɛrɛnkɛrɛnnen kɛ walasa ka sɛgɛsɛgɛli kelen-kelen bɛɛ furakɛ ni farikolokisɛ hakɛ kelen ye walasa ka batch nɔw bali. An ye diɲɛ farikoloɲɛnajɛfɛnw jateminɛcogo ɲuman kɛ, minnu bɔra ka tɛmɛ AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50) kan. Chromatographie sɛgɛsɛgɛli ni peak area integration kɛra ni MultiQuant version 2.1 porogaramu ye (Applied Biosystems SCIEX).
Iyɔn jate min ye 1000 ye, o kɛra sababu ye ka fɛnɲɛnamafagalanw nafa jateminɛ, wa TIC kɛra sɛgɛsɛgɛli kelen-kelen bɛɛ la walasa ka fɛnɲɛnamafagalan sɔrɔlen kelen-kelen bɛɛ ka yɔrɔ kuncɛ normalisé (normalisé peak area) jateminɛ walasa ka fɛn caman latilen minnu donna fɛn caman sɛgɛsɛgɛli fɛ minɛnw sɛgɛsɛgɛli fɛ ka bɔ sɛgɛsɛgɛli kɛcogo la. TIC normalizelen kɔfɛ, MetaboAnalystR(51) (paramètre défaut) bɛ kɛ ka logaritmi caman tigɛli kɛ ani ka norm line scaling automatique. An ye PCA kɛ ni vegan R pake ye walasa ka sɛgɛsɛgɛli kɛ metabolome danfara kan sample suguyaw ni ɲɔgɔn cɛ, ani ka baara kɛ ni partial redundancy analysis ye walasa ka banabagatɔw sɛgɛsɛgɛ. Aw bɛ Ward fɛɛrɛ kɛ ka funteni karti dendɔrɔgramme dɔ dilan walasa ka Euclide ka yɔrɔjan dalajɛ misaliw ni ɲɔgɔn cɛ. An ye limma (52) kɛ farikolojɔlifɛnw caya hakɛ dantigɛlen kan walasa ka farikolojɔlifɛnw caya danfaralenw dɔn selilɛri suguya bɛɛ kɔnɔ ani sigida fitininw bɛɛ kɔnɔ. Walasa ka ɲɛfɔli nɔgɔya, an bɛ baara kɛ ni kulu cɛmancɛ paramɛtiri ye walasa ka modɛli ɲɛfɔ, ka seli suguyaw jateminɛ sigida fitinin kɔnɔ i n’a fɔ kulu kelen-kelen bɛɛ (n = kulu 6) ka ɲɛsin nafa sɛgɛsɛgɛli ma, an ye sumanikɛlan saba segin-segin kɛ farikolojɔlifɛn kelen-kelen bɛɛ la Walasa ka an yɛrɛ tanga nkalon tigɛli ma, banabagatɔ donna i n’a fɔ balilan limma dilanni na. Walasa ka danfara minnu bɛ farikolojɔlifɛnw na banabagatɔw ni ɲɔgɔn cɛ, an ye olu lajɛ, an ye limma modɛli ladilan banabagatɔw sen bɛ o la cogo basigilen na. An bɛ danfara min ɲɛfɔra ka kɔn o ɲɛ, o nafa fɔli ye selilɛri suguya ni Padj ka sigida fitinin cɛ <0,05 (Benjamini-Hochberg ka latilenni).
Fanga sɔrɔli kɔfɛ ni Miltenyi Dead Cell Removal Kit ye ​​(>80% viability), selilu kelen transcriptome sequencing kɛra ascites congelées ɲɛnama bɛɛ lajɛlen kan ani tumor samples ni 10x 5′gene expression protocol ye. Ko duuru minnu ka kuru ni sɔgɔsɔgɔninjɛ bɛ bɛn ɲɔgɔn ma, olu sɛgɛsɛgɛra, hali ni ɲɛnamaya dɔgɔyali ka bɔ kuru kelen na, o y’a bali ka don a la. Walasa ka banabagatɔw sugandili caman sɔrɔ, an ye banabagatɔ kelen-kelen bɛɛ ka misaliw fara ɲɔgɔn kan 10x chrome controller siraw kan, ka asidi ni kuru yɔrɔw sɛgɛsɛgɛ u danma. Sɛgɛsɛgɛli kɔfɛ [Illumina HiSeq 4000 28×98 bp fila-fila laban (PE), Kebɛk jamu; kalansen 73 488 ani 41 378 ye selilu kelen kɔnɔ tumu ni sɔgɔsɔgɔninjɛ kama]], an ye CellSNP ni Vireo (53) kɛ (min sinsinnen bɛ CellSNP kan i n’a fɔ The common human SNP (VCF) provided by GRCh38 is assigned a donor identity.An bɛ baara kɛ ni SNPRelate ye walasa ka banabagatɔ ka genotype status (IBS) danfara surun (IBS) jateminɛ, ka fara seliluw ni seliluw kan minnu ma di u ma, minnu dɔnna ko duplexes ani donna minnu bɛ bɛn ascite ni tumor samples cɛ (54), an ye ko saba mara minnu ka cellules jiracogo ka ca tumu ni ascites kɔnɔ walasa ka jiginɛ sɛgɛsɛgɛli kɛ, an kɛlen kɔ ka mass filtration step kɛ scater (55) ani scran (56) BioConductor packaging kɔnɔ, o ye selilu 697 sɔrɔ (2792 and 4183 cells from tumor and ascites, respectively) for analysis. We use igraph's (57) Louvain clustering of shared nearest neighbor network (SNN) based on Jaccard distance to cluster cells by expression. The clusters were manually annotated to putative cell types based on marker gene expression and visualized with t-SNE. Cytotoxic T cells are ɲɛfɔlen don CD8A ni GZMA jirali fɛ, ka fara kulu fitininw kan minnu ka ribozomu poroteyini jirali ka dɔgɔn An ye Izar ni a ɲɔgɔnnaw ka kunnafonidilanw sɔrɔ minnu bɔra kɛnɛ kan (16), u ka t-SNE doncogo fana sen bɛ o la, o bɛ se ka jiracogo ɲɔgɔndan kɔlɔsi farikolo tangalanw taamasiɲɛw ni NNMT jirali cɛ.
PBMC farala ɲɔgɔn kan ka bɔ joli segin furaw la (STEMCELL Technologies) ni Ficoll gradient density centrifugation ye. CD3 + selilɛriw bɔra PBMC la ni CD3 biɲɛw ye (Miltenyi). Ni MNA bɛ yen walima ni a tɛ yen, CD3+ selilɛriw tun bɛ baara kɛ ni CD3 sirilen ye plati la (5μg/ml), CD28 min bɛ se ka wuli (3μg/ml) ani IL-2 (300 U/ml; Proleukin). Fɛnɲɛnɛma don laban na, ɲɛnamaya kɛcogo (Fixable Viability Dye eFluor450, eBioscience) ani a caya (123count eBeads, Thermo Fisher Scientific) jateminɛna ni flow cytometry ye. Aw bɛ kɛwalekɛlan baara jateminɛ ni aw ye farikolokisɛw lawuli ni PMA (20 ng/ml) ani ionomycine (1μg/ml) ye ni GolgiStop ye sanga 4 kɔnɔ, ani ka CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) ani TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD) kɔlɔsi. Aw bɛ qPCR ni ChIP selilɛriw lawuli ni PMA (20 ng/ml) ani ionomycine (1μg/ml) ye sanga 4 kɔnɔ. ELISA jilama lajɛra ka kɔn a lawulili ɲɛ ani a lawulili kɔfɛ ni PMA (20 ng/ml) ani ionomycine (1 μg/ml) ye sanga 4 kɔnɔ.
Aw bɛ a dilabaga ka sariya labato walasa ka ARN bɔ ɲɔgɔn na ni RNeasy Plus Mini Kit (QIAGEN) ye. Aw bɛ QIAshredder (QIAGEN) kɛ walasa ka sɛgɛsɛgɛli kɛcogo kɛ kelen ye. Aw bɛ baara kɛ ni ARN ka ADNc fɛɛrɛ ye min fanga ka bon (Thermo Fisher Scientific) walasa ka ADN dafalen (ADNc) dilan. Aw bɛ TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) kɛ ka jeninida jiracogo hakɛ jateminɛ (ka kɛɲɛ ni a dilabaga ka sariya ye) ni nin sonda ninnu ye: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogène (GAPDH)] ani Hs01010726_m1 (SLC22A2) ye. O sɛgɛsɛgɛliw kɛra StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) kan MicroAmp fast optical 96-well reaction plate (Applied Biosystems) kɔnɔ ni MicroAmp optical film ye. Ct hakɛ o hakɛ tɛmɛna 35 kan, o bɛ jate ko a bɛ sɔrɔ kɔlɔsili dancɛ sanfɛ, wa a bɛ taamasiyɛn kɛ ko a tɛ se ka dɔn.
Aw bɛ ChIP kɛ i n’a fɔ a ɲɛfɔra cogo min ka tɛmɛ (58). Kuma surun na, farikolokisɛw furakɛra ni fɔrmadehidi ye (a hakɛ laban ye 1,42%) ka sɔrɔ ka u bila so funteni na miniti 10 kɔnɔ. Aw bɛ funu tampon faralen ɲɔgɔn kan (25 mM Hepes, 1,5 mM MgCl2, 10 mM KCl ani 0,1% NP-40) kɛ jisuma kan miniti 10 kɔnɔ, o kɔ aw bɛ a lasegin farikolo tangalan tampon na i n’a fɔ a ɲɛfɔlen don cogo min (58 ). O kɔfɛ, sɛgɛsɛgɛli kɛra ni nin sɛgɛsɛgɛli ninnu ye: sɛrɛkili 10 (sekɔndi 1 wulicogo 20) ani waati jɔlen segin 40. Aw bɛ ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) ani SP1 (Cell Signaling Technology; 3μl) banakisɛfagalanw bila ka u lamaga ni u ye 4°CC lamaga su fɛ. Aw bɛ poroteyini A biɲɛw (Thermo Fisher Scientific) ni u ta kɛra 4°C la ni u lamaga dɔɔni dɔɔni fo lɛri 1, o kɔ aw bɛ chelex biɲɛw (Bio-Rad) kɛ ka ADN nafa, ani ka poroteyini K (Thermo Fisher) kɛ ka farikolojɔli dumuniw tobi. TNFα promotɛri sɔrɔla PCR fɛ: ɲɛfɛ, GGG TAT CCT TGA TGC TTG TGT; o kɔfɛ, GTG CCA ACA ACT GCC TTT ATA TG (207-bp fura). Ja ninnu dilanna Image Lab (Bio-Rad) fɛ, ka u hakɛ jateminɛ ni ImageJ porogaramu ye.
Selilu sɛnɛcogo sanfɛla lajɛra i n’a fɔ a ɲɛfɔlen don cogo min sanfɛ. O dantigɛli kɛra ka kɛɲɛ ni hadamaden ka TNFα ELISA minɛn (Invitrogen), hadamaden ka IL-2 ELISA minɛn (Invitrogen) ani hadamaden IFN-γ ELISA minɛn (Abcam) dilannikɛla ka fɛɛrɛw ye. Ka kɛɲɛ ni a dilannikɛla ka sariya ye, ji min bɛ sanfɛ, o bɛ kɛ 1:100 ye walasa ka TNFα ni IL-2 sɔrɔ, ani 1:3 walasa ka IFN-γ sɔrɔ. Aw bɛ EnVision 2104 Multilabel Reader (PerkinElmer) kɛ ka a minɛcogo suman ni 450 nm ye.
PBMC farala ɲɔgɔn kan ka bɔ joli segin furaw la (STEMCELL Technologies) ni Ficoll gradient density centrifugation ye. CD3 + selilɛriw bɔra PBMC la ni CD3 biɲɛw ye (Miltenyi). Ni MNA bɛ yen walima ni a tɛ yen, CD3+ selilɛriw kɛra baara ye ni CD3 sirilen ye plati la (5μg/ml), CD28 min bɛ se ka wuli (3μg/ml) ani IL-2 (300 U/ml; Proleukin) tile 3 kɔnɔ. Tile 3 kɔfɛ, aw bɛ seliluw lajɛ ka u ko ni saline 0,9% ye, ka sɔrɔ ka furakisɛ in kɛ ka a sumaya joona. Kɔnɔnatumuw jate kɛra ni flow cytometry ye (Cytek Aurora; 3L-16V-14B-8R configuration) ni 123count eBeads ye.
Aw bɛ farikolojɔlifɛnw bɔ i n’a fɔ a ɲɛfɔlen don cogo min sanfɛ. Aw bɛ furakisɛ jalen in labɛn kokura ni a hakɛ ye selilu 4000 ye/μl. Aw bɛ sɛgɛsɛgɛli kɛ ni sɛgɛsɛgɛli kɛcogo ye min bɛ wele ko reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) ani CORTECS T3 koloma (2,1×150 mm, a fɛnɲɛnamafagalan hakɛ 1,6-μm, pore hakɛ 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), o min kɔnɔ, ionisation électrospray bɛ baara kɛ cogo ɲuman na. Fasa mobili A ye asidi foromiki 0,1% ye (H2O la), fasa B ye 90% ye asetonitirili ye, asidi foromiki 0,1%. LC gradient ye miniti 0 fo miniti 2 ye 100% A la, miniti 2 ka se miniti 7,1 ma 99% B la, ani miniti 7,1 ka se miniti 8 ma 99% B la.O kɔfɛ, aw bɛ segin ka kolotuguda ni fasa mobili A balan ni jibɔ hakɛ ye mililitiri 0,6 ye miniti kɔnɔ miniti 3 kɔnɔ. . A jibɔ hakɛ ye mililitiri 0,4 ye miniti kɔnɔ, ka kolosinsinnan sumaya fo ka se 50°C ma. Baara kɛ ni MNA ka kemikɛli sariya saniyalen ye (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) walasa ka mara waati (RT) ni fɛn caman tigɛli sigi sen kan (RT = miniti 0,882, fɛn caman tigɛli 1 = 137→94,1, fɛn caman tigɛli 2 = 137→92 , Yɛlɛma 3 = 137→78). Ni 'tɛmɛsira saba bɛɛ Kɛra 'kɔnɔnafili waati bɛnnen na, 'tɛmɛsira 1 bɛ Kɛ hakɛ jateminɛni na walasa ka kɛrɛnkɛrɛnnenya Sɔrɔ. MNA (Toronto Research Chemical Company) ka kurufe jɔnjɔn kɛra ni stock solution (miligaramu 1/ml) dilution seriale wɔɔrɔ ye walasa ka standards sɔrɔ 0,1, 1,0, 10 ani 100 ng/ml ani 1,0 ani 10μg/ml jilama. Kɔlɔsili dan ye ng/ml 1 ye, ani jaabi sinsinni bɛ ng/ml 10 ni miligaramu 10μg/ml cɛ. Mikorolitiri fila pikiri kelen-kelen bɛɛ bɛ kɛ ka LC/MS sɛgɛsɛgɛli kɛ, ani ka jogoɲini sɛgɛsɛgɛli ɲagaminen dɔ kɛ pikiri 8 o pikiri 8 o 8 walasa ka sɛgɛsɛgɛli kɛyɔrɔ sabatili sabati. MNA furakɛlenw ka selilɛriw bɛɛ ka MNA jaabiw tun bɛ sɛgɛsɛgɛli kɛcogo sinsinni kɔnɔ. Kunnafoni sɛgɛsɛgɛli kɛra ni MassHunter quantitative analysis software (v9.0, Agilent) ye.
αFR-CAR sɛnɛcogo bɔnsɔn filanan bɔra Song et al. (59) ye. Kuma surun na, nin fɛn ninnu bɛ sɔrɔ o jɔli la: CD8a ɲɛmɔgɔ sinsinni, hadamaden αFR kɛrɛnkɛrɛnnen cakɛda kelen fɛn caman b’a la, CD8a hinge ni transmembrane region, CD27 intracellular domain ani CD3z intracellular domain. CAR sinsinni dafalen kɛra GenScript fɛ, o kɔfɛ, a kɛra clone ye ka don lentivirus expression vecteur bɔnsɔn filanan na GFP jiracogo kasɛti sanfɛ min bɛ kɛ ka transduction nafa jateminɛ.
Lentivirus bɛ sɔrɔ HEK293T selilɛriw jiginni fɛ [American Type Culture Collection (ATCC); min falen don Dulbecco ka Eagle medium modifié kɔnɔ min kɔnɔ 10% fetal bovine serum (FBS) ani 1% PenStrep bɛ sɔrɔ, ani ka baara kɛ ni CAR-GFP vector ye ani The packaging plasmids (psPAX2 ani pMD2.G, Addgene) bɛ lipofection amine (Sigma-Aldrich) kɛ. Banakisɛ bɛ ji min na, o lajɛra lɛri 48 ni lɛri 72 banakisɛfagalan kɔfɛ, ka a sama, ka a lajɛ ni ultracentrifugation ye. Aw bɛ banakisɛfagalan jilama caman mara -80°C la fo ka se a jiginni ma.
PBMC bɛ faranfasi ka bɔ joli-sira-funu-minɛn kɛnɛmanw na (STEMCELL Technologies) ni Ficoll gradient density centrifugation ye. Aw bɛ CD8 mikrobiɲɛw sugandili ɲumanw kɛ (Miltenyi) walasa ka CD8+ selilɛriw bɔ PBMC la. Aw bɛ T-cellw lawuli ni TransAct (Miltenyi) ye ani TexMACS medium kɔnɔ [Miltenyi; ka fara a kan ni hadamadenw ka joliseginfura 3% ye min ma baara kɛ ni funteni ye, PenStrep 1% ani IL-2 (300 U/ml)]. Lɛrɛ mugan ni naani wulilen kɔfɛ, T-cellw jiginna ni lentivirus ye (10 μl banakisɛfagalan min bɛ sɔrɔ a sanfɛ selilu 106 kan). Tile 1 fo tile 3 jiginni kɔfɛ Cytek Aurora kan (FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+ kan), aw bɛ GFP jirali jateminɛ farikolokisɛw la walasa ka jiginni nafa jira min bɛ se 30% ma
CAR-T selilɛriw ladonna sanga 24 kɔnɔ Immunocult (STEMCELL Technologies; faralen don a kan ni 1% PenStrep ye) nin cogoya ninnu na: u ma furakɛ, u furakɛra ni adenosini 250 μM ye walima ni MNA 10 mM ye. Furakɛli ɲɛfɛ, CAR-T selilɛriw ko ni PBS ye ka u fara ɲɔgɔn kan ni SK-OV-3 selilɛri 20.000 ye [ATCC; McCoy 5A medium (Sigma-Aldrich) la min farala FBS 10% kan ani PenStrep 1% la 10 la: Effecteur ni target ratio 1 cayara siɲɛ saba Immunocult medium faralen kan. SK-OV-3 seliluw ni SK-OV-3 selilu minnu wulilen don ni digitalis saponin ye (0,5mg/ml; Sigma-Aldrich) kɛra kɔrɔsili juguw ni kɔrɔsili ɲumanw ye. Lɛrɛ 24 kɔfɛ, u kɛlen kɔ ka sɛnɛ kɛ ɲɔgɔn fɛ, ji min bɛ sanfɛ, o lajɛra ka lactate dehydrogenase (LDH) suman ka kɛɲɛ ni a dilabaga ka laadilikanw ye (LDH Glo Cytotoxicity Assay Kit, Promega). LDH ji min bɛ sanfɛ, o bɛ dilan 1:50 LDH tampon na. Kɛmɛsarada la, mɔgɔfaga hakɛ jateminɛna ni nin formula in ye : mɔgɔfaga kɛmɛsarada la = latilenni kɛmɛsarada la / mɔgɔfaga hakɛ maksimali x 100%, yɔrɔ min na latilenni kɛmɛsarada la = co-culture-T cellules dɔrɔn, ani fagali hakɛ maksimali = control positive-negative control.
I n’a fɔ a ɲɛfɔlen don cogo min na sɛbɛnni kɔnɔ walima fɛnw ni fɛɛrɛw kɔnɔ, aw bɛ baara kɛ ni GraphPad Prism 8, Microsoft Excel walima R v3.6.0 ye walasa ka jateminɛ kɛ. Ni sɛgɛsɛgɛli caman kɛra banabagatɔ kelen fɛ (i n’a fɔ sɔgɔsɔgɔninjɛ ni tumu), an bɛ baara kɛ ni t sɛgɛsɛgɛli fila-fila ye walima ka banabagatɔ don a la i n’a fɔ nɔ gansan dɔ modeli sinsinni walima misali caman na i n’a fɔ a bɛnnen don cogo min na. Walasa ka metabolomiki sɛgɛsɛgɛli kɛ, nafa sɛgɛsɛgɛli bɛ kɛ siɲɛ saba.
Walasa ka kunnafoni wɛrɛw sɔrɔ nin barokun in kan, aw bɛ se ka http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1 lajɛ
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Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zakari (Lauren G. Zakariya), Abigayɛli Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. Deberardini), Russell G. Jones (Russell G. Jones), Phineas T. Hamilton (Finɛsi T.) ye baara kɛ ni a ye.
MNA bɛ dɛmɛ don farikolo T-cellw ka farikolo tangacogo la, wa a bɛ farikolo tangacogo laɲini dɔ jira min bɛ se ka kɛ hadamadenw ka kansɛri furakɛli la.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zakari (Lauren G. Zakariya), Abigayɛli Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. Deberardini), Russell G. Jones (Russell G. Jones), Phineas T. Hamilton (Finɛsi T.) ye baara kɛ ni a ye.
MNA bɛ dɛmɛ don farikolo T-cellw ka farikolo tangacogo la, wa a bɛ farikolo tangacogo laɲini dɔ jira min bɛ se ka kɛ hadamadenw ka kansɛri furakɛli la.
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