Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. grWo Kam Ngar-Woon carcinoma nasopharyngeal immune in refractory response rescues cisplatin with combined Idronoxil Guan eiti,dizi n lcti.Irnxl(D,as nw speooil sasnhtcdrvtv of derivative causes IDX synthetic that a shown have is studies predominately phenoxodiol) Preclinical comprise as 1995). of known which Nelson, risk also isoflavones & Waring the (IDX, products soy Reeder, Soy in Idronoxil Kelly, including (Joannou, reduction 2005). phytochemicals, glycitein. genistein modest Spitznagel, soy and & a of daidzein Yan including sources genistein, 2006; benefits, Clarke, main health & the Hilakivi-Clarke many provide histological (Trock, with (TME). commonest cancers microenvironment associated certain tumor (the and the is developing NPC into infection soy undifferentiated cells (EBV) immune of of of virus Consumption hallmark infiltration Epstein-Barr substantial a The between is Moreover, 2014). regions) association al., endemic III. high in et type type (Sun the treatment especially is intensive radical risk, cancer Unfortunately, this NPC this 2017). despite al., years of et 5 characteristic Shield ad- within main 2015; or relapse al., still chemoradiation) et patients (concurrent (Blanchard of additional diseases 30-50% advanced with NPC therapy, with locally Patients for radiation chemotherapy 2006). high-dose junct Adami, with & (Chang treated China uniformly Southern are in endemic is (NPC) carcinoma Nasopharyngeal Introduction with combination text in Main and responses, immunomodulatory expression). induce PD1 to and IDX CD62L immunity. of (attenuated antitumor abilities potential enhance functional chemotherapies, the conventional and underscore IDX where homing findings axis, tissue Our (IFN) increased interferon Conclusions. of I CXCL10/type acquisition the we with Mechanistically, by spheroids mediated cells. tumor largely T IFN was (DP) in cells double-positive elevation T and an DP cancer CD8+ precipitates in migratory CD4+, cisplatin in of and increase priming IDX the and of that treatment trafficking showed combination expansion, importantly, the More enhance cytotoxicity. mice can and in (1-2 subcutaneous cells proliferation tumors in IDX cell cells nasopharyngeal T of T promotes on human low-dose infiltrating also toward (IDX) evaluate of but cells with to Administration idronoxil lines T out cell carried of Results. of were NPC function analyses effects of models. and immunohistochemical cocultures the and phenotype spheroids cytometric activation, 3D examined Flow and infiltration, we monolayer EBV-positive vitro. the 2D authentic assess Here, analyzed in We to tested cells NPC43). was disease. and immune IDX C17 and/or this (C666, cisplatin treating lines of cell activity in NPC-derived antitumor How- The challenge infiltration. Methods major lymphocytic interactions. prominent a tumor-lymphocyte by remains characterized cancer recurrence chemosensitive ever, a is carcinoma Nasopharyngeal Background. Abstract 2020 12, August 3 2 1 Kwong h nvriyo ogKong Hong of University The Limited Noxopharm Kong Hong of University 1 lve Laczka Olivier , 1 1 emn a-agHung Tae-Yang Desmond , 2 α onWilkinson John , eesadlast XL0idcin diinly D nacdTcl nlrto towards infiltration T-cell enhanced IDX Additionally, induction. CXCL10 to leads and levels 2 a-a Tsao Sai-Wah , μ )ntol estzscne el oceohrp i apoptosis, via chemotherapy to cells cancer sensitizes only not M) 1 1 a i Yim Kin Man , 3 itrH-u Lee Ho-Fun Victor , 1 aCu Wu Chun Ka , 1 1 Xin-Yuan , n Dora and , Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. etfrls hn4wesa -20 solution) at (stock weeks mg/mL 4 40 [?]0.1%. at than DMSO less in for suspended kept –80 was at Australia) were stored Limited, and Tissues Noxopharm IRB, Review by HKW Institutional (HKU/HA (provided Kong. the Cluster IDX by West Hong approved Kong of Hong been 4 Authority University has were Kong/Hospital and study Hong the study UW16-428). P/S The of this Hospital, University % in examination. The Mary of used histological 1 Tsao Board Queen samples to FBS, tissue George subjected to % NPC and Professor admitted 10 fixed by ALX-270-333). patients with provided Sciences,. from supplemented kindly Life obtained (Invitrogen) (Enzo NPC43+ve), inhibitor medium and ROCK NPC43-ve RPMI1640 Y27632 C666, with (C17, cultured conventional lines were to cell cells NPC Four NPC of reagents and response tissues poor Cells, antitumor the evoking be and by may methods indirectly, TME IDX and Additionally, also Materials concerning and insights tumors. directly further within both migration gather chemotherapy. and cells to into cancer tool infiltration kills a T-cell markers, IDX dysfunctional modulating Thus, and by homing immunity node PD-1. (DP) lymphoid double-positive both and of for CD62L expression enriched downregulated human were demonstrated towards spheroids cells cells these DP IDX-treated of in functions infiltrates and activation cell CD4 infiltration, immune the study and Increased to IDX functions anti-tumorigenic cells both tumors. immune cytotoxic of elicit and augmented combination cells and CD8 alone, with NPC regulating impact TME of in migration significant synergistic the show T-cell was targeting not Our induce re-educate cis did and could treatment to treatment. IDX itself (cis) IDX While IDX cisplatin capacity. of capacity, with proliferative ability this conjunction In purported in migration a effects. and demonstrated proliferation also the results inhibit and proliferation cancer apoptosis IC and cells to the TME T the determined and controls we IDX activity Here which cell by its mechanisms NK exact through of the unclear. immunomodulator augmentation tumors, remain an CD56 of the progression the as treatment Despite IDX the specifically for classified system, 2009). critical Ritchie, immune also being al., have & the et Berridge of al. (Georgaki arm Neeson, et innate Davis, population Georgaki the apoptotic (Herst, and the stimulate cells IDX targeting to T for be capacity activated target may IDX described a and that as report illustrate proliferation 2009) one (Yagiz, findings fibroblasts These Indeed, the IDX embryonic Moreover, 2007). mouse itself. inhibit Morre, immortalised machinery 2006). to & in bioavailability al., are Morre genes low reported et James (IFN)-stimulated issue (Gamble Kuntz, interferon its been Wu, this of angiogenesis of range overcome previously inhibiting a thereby result to has induce cells, can strategies a IDX endothelial though as human Strikingly, orally, discontinued of or migration was explored. intravenously journey either being IDX’s administered currently past, when the (Aguero, humans cells in in cancer tested in damage clinically DNA binding Whilst promote The and oxidase arrest 2005). 2007). dinucleotide cycle Senderowicz, Morre, adenine cell & & induce Ecto-Nicotinamide Sheleg Kim can as Facchinetti, Balicki, protein receptors Yagiz, known a respective Chueh, is to cells IDX (Morre, IDX cancer of (ENOX2) of mechanisms all target 2 pro-survival Another of type also cell exchanger PI3 membrane 2012). is regulating disulfide-thiol and al., outer IDX kinase for et al., the Yao sphingosine et responsible 2020; on genistein. including (Alvero and Wilkinson, found enzymes than chemotherapy & cells of effective Delebecque to inhibition cancer Laczka, more cancer its Fairlie, in to (Porter, of times over-expressed related types 5-20 are likely certain which is is IDX sensitize kinase and of can lines activity which The cell models 2008). cancer cancer of murine range in wide active a in apoptosis + CD8 + el,mdae yteu-euae xrsino IFN of expression up-regulated the by mediated cells, ° .Sra iuin ftesokslto eefehypeae ncmlt eimand medium complete in prepared freshly were solution stock the of dilutions Serial C. 50 fIXo P ellnsadsoe hti ol ute estz h cells the sensitize further could it that showed and lines cell NPC on IDX of ° .I l utrs h nlcnetaino MOi h eimwas medium the in DMSO of concentration final the cultures, all In C. + elepnin enx eeoe peodcocultures spheroid developed next We expansion. cell T 2 α/ XL0 atclry h infiltrated the Particularly, CXCL10. + aua ilr(K cell (NK) killer natural μ M Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. FEpstv el nTcl outr el n aa eldahfo el akn outrdTcells. % T - cocultured of death lacking staining wells dye cell from viability specific death from (% cell determined x basal was 100 and death wells = cell coculture Specific lysis specific cell specific of death. 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. 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D nacstecttxct fcsltnaantNCcl lines cell NPC against cisplatin of cytotoxicity the enhances IDX comparing when post-test groups Dunn’s Results 2 with comparing Kruskal-Wallis when value test or p ANOVA U A two-way Mann–Whitney appropriate. the the by by analyzed were and variables quantitative in Differences expression suppressed or to at lead found might Statistics CpG usually of are methylation sites [HG- of CpG GPL570 level genes. high transcripts. of the so RNA sites (TSS), on of CpG normal site targeted based 3 start probes transcription were and the near specimens datasets and NPC BeadChip The GSE53819 450K 13 microdissection. Methylation tissues, included laser GSE34573 nasopharyngeal whereas by normal U133 specimens excised 10 normal tissues and 18 Biotechnology nasopharyngeal and specimens for expression NPC NPC Center gene 18 31 National the included from The contained scale. obtained of GSE12452 color were GSE53819 database by Information. GSE52068 GSE34573, https://www.ncbi.nlm.nih.gov/geo/) datasets indicated datasets profiling was (GEO, profiling methylation expression expression omnibus gene ENOX2 gene and NPC patients. plotly. GSE12452, 11 by and from generated obtained were expression visualizations were ENOX2 for KRT19. Data used cells and were KRT15 biopsies tumor KRT5, normal EPCAM, malignant 1 using and identified in tumor were NPC cells 15 Epithelial from analysis. Data global GSE150430. from obtained were data Single-cell collection 37 at data microarray medium har- and digestion cell were in Single Tumors enzymatic incubated were and Tissues time. mechanical indicated (Sigma). mechanically both I were the 1.5 required DNase cells 20 at leukocytes using Spleen for days mg/kg, tumor-infiltrating by day staining. 10 2 every of digestion immunohistochemical IDX every injected isolation for with monitored were sections whereas injected Drugs was tissue disaggregated, were intraperitoneally. growth paraffin mice Tumor cis prepare inoculated and growing, to were weeks. IDX vested start group of three to combinations per to tumors or mice up the mg/kg) nude expect differ- (5 Thirty we Cis cells Researchmg/kg, when T retained. extrathymic and 10, were Teaching functional day cells 10 but in Around x T 5 developed, Animals of with not 8-weeks-old Live levels subcutaneously were in once low of performed cells and were Use T Experiments pathways, thymus-derived the entiation 4592-18). which on (protocol in Committee Kong mice, Hong the nude of by University the approved of was (CULATR) work mouse All experiments tumor vivo In with visualized instructions. and kit’s DAKO) mouse the (K5007, (DAKO) System to with Detection reagent according incubated EnVision (DAB) 4 blocking then REAL diaminobenzidine DAKO at were peroxidase with overnight Sections performed with ab210751) was X0909). blocked 95 staining abcam (DAKO, at was (1:500, block heating peroxidase ENOX2 protein by anti-human Endogenous binding step non-specific retrieval by antigen (pH6). followed an buffer by citrate followed in rehydrated, xylene, in deparaffinized and 4 duplicate of in Sections analyzed were samples reported. All were Immunohistochemistry GAPDH. runs to Ct 2 Comparative values the the Ct of using mean the measured the was normalizing quantification and Relative Cycle) Roche. (Threshold LightCycler480 a on (TaKaRa) Mix Plus ]AyerxHmnGnm 13Pu . ra ltom S508ue nnu Human Infinium used GSE52068 platform. Array 2.0 Plus U133 Genome Human Affymetrix 2] μ hcns eectfo omlnfie aanebde isebok.Tesie were slides The blocks. tissue paraffin-embedded formalin-fixed from cut were thickness m < μ /LclaeaeI Rce,08m/Ldsae(nirgn,ad01mg/mL 0.1 and (Invitrogen), dispase mg/mL 0.8 (Roche), IV collagenase g/mL .5wscniee ssaitclysignificant. statistically as considered was 0.05 6 P ellns+mtie 11 sn 9guenel ne anaesthesia. under needle 29-gauge a using (1:1) matrigel + lines cell NPC 5 o .Sie eete ahd n secondary and washed, then were Slides C. ° o 0min. 30 for C > ruswhen groups 2 o o 5min 45 for C Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. 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IDX pronounced The EBV-negative demonstrated D). killing and cells in increased effective 1C NPC43-ve dose-dependently (Figure IDX, IDX to control. Likewise, contrast IC untreated compared 1B). the migration with (Figure cytotoxicity with reduced manner diverse significantly compared showed dose-dependent cells IDX a apoptotic with in late-stage treated cells, We cells untreated Cancer B). with and apoptosis. 1-4 and 1A from migration (IC IC Figure ranging with viability (Supplementary doses hours cell 120 expression data, 50% over cells ENOX2 of C666 inhibition for and 2-4 caused C17 positive NPC43+ve, that NPC43-ve, all NPC43- concentrations of (NPC43+ve/ were drug proliferation cells the study NPC determine EBV-positive/negative this and first C666 in C17, used lines cell ve) NPC parental human The μ μ Fgr A;hwvr P4-esoe ihrrssac oIXtetet ae nti initial this on Based treatment. 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(1.32-fold; that et treatment and to Muthuswamy combination cells, compared IDX2+cis1.2 tumor 2011; as and of 4G) (1.5-fold) al., supernatant IDX2 Figure the et by protein in influenced (Berghuis present in particularly was 2D) was increase CXCL10 regulation that Figure 2.93-fold that demonstrated involved Supplementary a further observed be We demonstrated DMSO; we to (densitometries 2012). to Notably, known tumor CXCL10, compared 2014). suppress into component as al., downstream and recruitment expression I et cleave chemotactic IFN White cells potentially of 2019; T to production reported al., in shown the previously et induced was those (Ning greatly caspase-7 with alone production consistent IDX2 where IFN is functions I finding cascade type This caspase STING-mediated alone. apoptotic IDX opposite the and were control on cascade to caspase-7 compared apoptotic as treatment, of IFN- patterns IFN- This of treatment. protein detection those IDX IFN the to and I control Interestingly, type to and the compared treatment. pathway for as combination observed lines production cell also IFN NPC was IRF3 in I effect downregulated Ser386, suppressive cis type We not of but cis1.2. the administration Ser396, by IDX1. impairing at expected, extent, In phosphorylation with As expression, pathway. capacity, greater production. sustained STING a signalling repair chemokine levels to compromise proinflammatory (STING) DNA cancer with and suppressing may genes lines, defective treatment, in cis IDX2 cell IFN with signaling that upon NPC of cells speculate reduced downstream in stimulator to unexpectedly, expressed on the was, restricted constitutively by expression combination was be STING modulated protein in to likely STING or suggested and 4F, been alone Figure cells has cancer cis IDX as IDX, of such of action Figure effect The Supplementary the 4C-E, cells. (Figure investigated site next tumor combination We towards that cells suggest T results DP These of DMSO. migration 2C). to the favor compared could when treatment IDX2+cis1.2) and (IDX1+cis1.2 ments CD4 CD4 36.66% enhance (mean dose-dependently could cell cells tumor to CD8 alone IDX of CD8 Similarly, exposure and 4A). subpopulations, Figure cells IDX2+cis1.2- T p=0.0052; towards paring (2.4-fold, PBMCs control % CD3 migrating (53.03 DMSO migratory of IDX2+cis1.2) to of number number compared total increased observed the an were in response cells increases in players cancer PBMCs Substantial critical of treated capacity most treatment. migratory the the drug amongst investigated next are to we mediators therefore, cytotoxic tumorigenesis; produce resolving toward can that cells immune capability Tumor-infiltrating migratory killing. enhanced T-cell-mediated with axis the to cells IFN confirming susceptible T ability, I endows more killing cis tumor increased with the an combined making acquiring IDX mediate in toward directly treatments cells only T these not combination IDX1/IDX2+cis1.2- prime of in also role by or can alone critical treatment educated but (Figure cis manner apoptosis, previously and ratio-dependent cell IDX effector-to-target cells. were that tumor an primed-T indicate in which clearly enriched cells data achieve tumor cells These killing to 3E). of primed-T IL-2 capable were assay, with cells expanded cancer cytotoxicity treated further next the and we post-cocultured on collected induction, PBMCs cell were Based T functions. cells drive effector cancer could elicit could cells treated cells cancer with tumor-primed-T in the IDX+cis whether or determine alone to IDX sought with treatment 3D). that shown (Figure Having IDX2+Cis1.2 induced and significantly IDX2+Cis1.2 IDX1 and which with alone treatment IDX2, IDX2 expressing upon with with cells CD8 treatment treated on that of were focused illustrates frequency then 3C cells the We Figure 3B). when (Figure CD8. restored treatment and combined was CD4 IDX+cis effect upon upregulated This further was diminished. substantially were subsets + + CD8 el(en61.2% (mean cell T + + elmgain(upeetr iue2) atclry h nrae rprino migratory of proportion increased the Particularly, 2B). Figure (Supplementary migration cell elsbes(en78.39% (mean subsets cell T α, hr lae aps- a on ob ral nrae nNCcl ie olwn cis following lines cell NPC in increased greatly be to found was caspase-7 cleaved where ± .4 n 33.5% and 9.446 + el scmae oDS.Seicly D/D ai ntecclue increased cocultures the in ratio CD8/CD4 Specifically, DMSO. to compared as cells ± .5,p003 n 81 % 68.18 and p=0.0236 8.859, ± .3 n 64.99% and 1.937 ± + .7 epciey,btmrel oe hndul-oiie(DP) double-positive than lower markedly but respectively), 3.87, ± el eeosre ncmiaintetet IX+i12and (IDX1+cis1.2 treatments combination in observed were cells 18 n 82.19% and 11.81 ± .3,rsetvl)wssihl rae hnCD4 than greater slightly was respectively) 8.539, 7 ± .9,p002,rsetvl;Fgr B.Com- 4B). Figure respectively; p=0.0029, 7.299, ± 54,rsetvl)uo obnto treat- combination upon respectively) 15.48, α, hl t xrsinwsrsoe upon restored was expression its while via CXCL10-type a + T + Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. o D,C6LadP1uo ramn ihIXi h outrs otcnclyvldt u findings, our validate technically To cocultures. expressions the activation panel surface in and IDX cell different CCR7) with T substantial treatment and the observed upon (CD62L explored we PD1 we receptors biomarkers, and homing those CD62L charac- Among node CD3, were for PD1). cell lymph subsets T na¨ıve and for (CD45RA) 6A). and (CD27 gated Figure (CD45RO) markers thereafter (pink, sub- dissimilar memory control were cell observed by to We which T followed compared cytometry. terized as determined flow CD8), (blue) we through CD4, treatment markers treatment, (CD3, IDX surface IDX following phenotypes cell cells upon 10 infiltrating spheroids of of the patterns patterns expression infiltrate on cells based T sets particular which cells T assess in To markers homing and activation thereby of chemoattractants, expression infiltrates T-cell differential of induces killing. expression administration tumor the IDX further induces augmenting also possibly but growth, infiltration, com- conclusions spheroid tumor the T-cell previous tumor further, in arrest our enhancing extent, refine only explore the lesser experiments not To to a these IDX 4G). Collectively, to added that spheroids. also, (Figure were tumor and hypothesis towards CXCL10, migration, treatment, this PBMC chemoattractants, bination IDX-induced expres- supports the reduced the 10 significantly of in which and resulting the one cultures, IDX 9 infiltrate to against CXCL8, lines turn, antibodies chemokines cell in NPC neutralizing T-cell that of the Exposure PBMCs gradients. of activate chemokine might sion of cells influence the tumor under IDX-conditioned tumor that hypothesized next control We to tumor compared CD107a in of process 3B). modest, increase; apoptosis Figure albeit previous of levels, (1.4-fold active (Supplementary our increased induction an alone. with showed the spheroids agreement to cis confirm tumor In proportional IDX2+cis1.4-treated to to F). was coculture compared 5D, infiltration the (Figure when spheroid on 75.5 cells that higher staining (mean observed CellEvent was PBMCs and performed hours infiltrated also apoptosis where 72 we tumor Moreover, incubation, infiltrated after cis-treated 5D-E). of elevated (DMSO) Figure in hours spheroids in infiltrated 24 control PBMCs resulted after Strikingly, the decline treatment increase. to Combination 3.1-fold started spheroids (p=0.0149) alone. PBMC of significant of hours cis a influx 72 representing to over the 164.5 (DMSO) increased compared controls significantly (mean when the treatment PBMCs combination in spheroids early constant and as remained the alone detected IDX cells was into that infiltrated treatment (OUT) found of upon medium We infiltrated number spheroids the tumor the coculture. within in the while remaining in composition hours, infiltration cells 2 cellular PBMCs and as in the (IN) were increase that cells studied An donors infiltrating 5C). cocultures. we healthy separating (Figure from heterotypic mechanically coculture, obtained by through After PBMCs spheroid PBMCs with tumor cocultured and possible. line spheroids whenever cell tumor NPC HLA-matched an between from interactions generated was the Spheroids “CellEvent,” explored 3D next the We in fluorescent effect IC using the IC drug determine the apoptosis The to Overall, on curve 5B). imaging. dose-response panel) a spheroid right bright-field build con- real-time with to 5A, increasing combination used persistent (Figure in of Red) perform cis Deep effect to LysoTracker and and the microscopy panel) Green monitored Caspase-3/7 left CellEvent We 5A, (non-toxic 3A). probes (Figure Figure IDX (Supplementary of cultures centrations tumor (3D) dimensional line- cell mimic cancer To nasopharyngeal treatment in IDX either cells upon IDX, tumor-infiltrating spheroids with and derived treatment apoptosis upon cell tumor augmented T Increasing significantly cis1.2. in be involved with [1.26-fold 3.45-fold]) combination to CXCL9 and in found 1.17-fold], [3.66-fold or were alone and TNFRSF1A chemotaxis and [1-fold and 1.24-fold], CCL8 activation and cell respectively], [1.48-fold IDX2+cis TNFSF1B and 1.18-fold], IDX2 and 2.11-fold, and [1.93-fold nvivo in odtos eeaie h yooiiyo D n i nNCclsuigthree- using cells NPC on cis and IDX of cytotoxicity the examined we conditions, 50 ± auswr oprbebtenteshri-eie el n3 n Dconditions. 2D and 3D in cells spheroid-derived the between comparable were values .)wti h peod fe 2hus oprdt i ln ma 53.0 (mean alone cis to compared hours, 72 after spheroids the within 4.5) via uoai aigb efrigmliait lseigo el ae na on based cells of clustering multivariate performing by gating automatic 8 nvitro in 50 ausfrcsa 1.4 at cis for values eut Fgr D,teifitae BC in PBMCs infiltrated the 2D), (Figure results μ n D t2.1 at IDX and M ± μ (Figure M 5 within .5) ± 6.0), Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. uemc r rsl eceti eihrlTcls h ubro ypoye ern -elmarkers while T-cell model, bearing our in lymphocytes Analogously, of 1992). athymic number Lake, Although & the B). Pierce cells, (Kennedy, 7A, T age (Figure with peripheral CD14 group steadily in increases control deficient CD8) the grossly (CD4, 5mg/kg to are and (IDX20+cis5) compared 20mg/kg mice combination-treatment and nude growth, 10 nude high in of tumor that tumors dosage decreased a demonstrated NPC43+ve at curves nasopharyngeal significantly cis growth of and IDX growth The with the respectively. every-other-day weight/day, exhaustion on treated were IDX weak animals of a test effects mice, with antitumor spheroids possible evaluate infiltrate To to prone are vivo cells In 62.10 T (mean memory treatment. IDX DMSO DP upon to that PD1 profile compared in suggest as up-regulation results significant treatment These found IDX PD1 we 48.57 in upon (mean contrast, reduction cells controls In significant DMSO a 6E). to detected Figure compared we 0.0086; c), cells 6A, memory (Figure infiltrating findings total above the to additionally muemdae etuto nNCtmrcls hl digcsi obnto ol ute enhance and further tumor-killing could combination a Conclusions in elicit and cis Discussion potentially adding could while treatment be cells, as IDX might tumor effect. methylation thus activity this NPC DNA ENOX2 and antitumor-responsive in decreased cancer, the with destruction such, NPC immune-mediated associated As with was 7G). which associated higher (Figure significantly tissues, dataset closely a normal GSE52068 at illustrated expressed the to GSE34573) and in compared (I–IV) and evident progression as cancer GSE53819 (Figure tumors of staining (GSE12452, stages in sequencing histological all datasets levels at by available present GEO level was publicly single-cell protein gene microarray at ENOX2 a at ENOX2 publicly that From confirmed expressed three further type NPC. was Another cell in main feature 7G). the effect this were 7E-F), anti-cancer cells (Figure malignant pronounced resolution that IDX’s ENOX2 observed of we to expression GSE150430, the link CD4 investigating cohort by potential infiltrating findings signature a of our of as percentage relevance clinical the the evaluated in we Finally, (Figure differences group significant DMSO no groups. the were all in 2.860 there among observed 7C; However, that (Figure 4D). than group Figure mice cis IDX20+cis-treated the 1.668 in 7D; in higher significantly detected was that TILs, CD8 than infiltrated mice of IDX20+Cis-treated 8.482 number of higher tumor significantly the observed CD4 we for (TILs), treatment drug after CD8 and before 4A-C; CD8 CD45RO of proportions decreased CD45RO IDX showed following for (Figure infiltration cells control spheroid toward DP T to advantage CD3/CD4/CD8 compared tumor-infiltrating particular DP on Additionally, expression a analysis have CCR7, 6B). gating could not Through (Figure cells but treatment. T treatment CD62L, DP IDX2 in that reduction upon suggesting pronounced 6C), structure a tumor displayed the cells T in cells T DP cocultured c). CD3 6A, in comparing PD1 (Figure PD1 By upregulated per- expression that of by CD45RO such Surprisingly, expression observed, reduced b). was fluctuated to correlation 6A, correlated observed inverse (Figure an expression we Interestingly, expression treatment. SOM, CD62L IDX2 generating in upon PBMCs and up-regulation reduction an and clustering illustrated a), k-means clusters 6A, forming the (Figure of expression rose-charts CD3 Representative of algorithm. (SOM) map self-organized + + ± + el a 4.971 was cells T el eeas eetbei h iclto cosaleprmna rus SplmnayFigure (Supplementary groups. experimental all across circulation the in detectable also were cells T .5,rsetvl) motnl,tepretgso CD8 of percentages the Importantly, respectively). 1.758, nitmreeti soitdwt nrae el infiltrations cells T increased with associated is effect anti-tumor mncts n CD19 and (monocytes) ± .10ad5.400 and 0.5130 - CD27 + + aı ecls scmae ocnrl(iue6,SplmnayFgr C.Finally, 3C). Figure Supplementary 6D, (Figure control to compared as na¨ıve cells, elppltosI n U fteshris eosre infiatices in increase significant a observed we spheroids, the of OUT and IN populations cell ± .9 n 9.675 and 1.390 ± .2,rsetvl) hc a ofimdb muotiig(Supplementary immunostaining by confirmed was which respectively), 1.426, + Bcls ypoye ersne stemi w ust,CD4 subsets, two main the as represented lymphocytes cells) (B ± .1,rsetvl) nrlto otmrifitaiglymphocytes tumor-infiltrating to relation In respectively). 5.213, + el a 19.47 was cells T 9 + + el olwn D-rae outr,infiltrating coculture, IDX-treated following cells T CD27 + + ± el,abi tlwlvl mn l live all among levels low at albeit cells, T .4 s en43.57 mean vs. 7.049 eoyclsadicesdproportions increased and cells memory + ± .7 n 18.01 and 5.877 el ihnCD19 within cells T ± .1 s en62.06 mean vs. 5.117 + xrsini na¨ıve infiltrated in expression ± ± .1,respectively; 5.411, .7;p=0.0017). 3.870; - + ouainin population xrsinin expression ± ± .6;p= p 3.066; .4 and 1.548 + cells T + and Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. efrhrdvlpdhtrtpccclue fNCtmrshriswt el osuyteinfiltration, CD4 DP the activated/memory study that to showed cells study T this established. with Critically, be spheroids infiltrates. to tumor of remain NPC mtDNA function of inducing and by cocultures phenotype the cells heterotypic through to NPC developed contribute functions the further might their on We activity damage has STING mitochondrial exert cell in upstream cis apoptotic of perturbation and an result whether impact the and IDX IFN immunological mechanism, is action, in chemoattractant signaling of alternative elevation of induction an the mode the or trigger Whether the to to leading to likely pathways. cascade comes more caspase signaling modestly was it downstream IDX cis when apoptotic that while least observed different IFN shown), inhibit we at to not Likewise, back that, fed data latter 2014). highlights (mtDNA, the al., and DNA et caspase-7, As mitochondrial apoptosis apoptotic (White cleaved during impaired. of IFN functions partially release cascade producing was the IFN caspase pathway from at sustaining induced STING cells pointing of discovery the dying recent capable prevent that a CD8 is toward given to attention IDX of cells our why NPC trafficking drew to we in and such, as expressions cells CXCL10, question CXCL10 of antigen-presenting important of expressions of an regulation the homing raised Accordingly, This the cells. we NPC for attenuated. Herein, in chemokine treatment attention. important cis gain following an observed to only Unexpectedly, starting IDX was IFN lines. higher is CXCL10, cell in whilst NPC response expression, reduction in STING antitumor protein strong in endogenous STING the a STING down-regulated on cisplatin impact in of with no combination role play had in the IDX may to concentrations of effects, potential it field concentrations antitumor low the role STING promotes that has the observed the clearly The which and compartment date, response myeloid To cells, IRF3 T-cells. the tumor predominant upon in study. of a extent STING CD8 this activates migration of and larger in it activation and cells a that IFN/CXCL10-dependent activation dendritic concept to I recruit the expansion, and type help on IDX, the partially focused with drives be been tumors during to has cis, In suppressed of thought and significance. often conditioning is of are IDX that effect is and of observed interactions response treatment we immune tumor-lymphocyte antitumor assay, modulates combination the positively coculture of IDX 2D effectors that our main finding the our activate of disease, to one This the aiming are ability. interventions cells cytotoxic therapeutic T their future As unleash in to agent cells this T of lymphocytes. proliferating application tumor-reactive allowing the by supports effects further anticancer finding its to (4 phase findings concentrations addition previous high in in at confirmed cancers. IDX we proliferation tested whilst metastatic Herein, cell that Ritchie, studies. and was demonstrated T these & primary and in and Berridge 2009) of optimised al., Neeson, FDA fully types et Davis, not (Georgaki various the was (Herst, human against dosing use from that past 2006) phenoxodiol in suggesting approval a Alvero, with 2009), interest & track during associated Our Fu was obtained fast (Mor, lymphocytopenia drug. results However, humans gained from chemotherapeutic in phenoxodiol part a trials in 2004, with II/III stems In combination IDX was function IC study. in that immune IDX showing of clinical impacts apoptosis Although findings range IDX lines. previous to how restricted confirmed cell cells we determining relative cancer Additionally, these the lines. NPC sensitizes cell cis, of cancer further range than various treatment. the a cytotoxic cancer across against less of response IDX foundation be future of new the to cytotoxicity a to found the contributing lay at determined therefore chance might first second We IDX a of molecule involved properties unique pathways elucidating immune-stimulating activation this effect, appropriate the The giving anti-tumor seeking toward the disease. finally, its and to NPC determining into interactions immuno- addition fighting finding: trafficking tumor-lymphocyte in in drugs on and this response immune IDX the immune priming allow of an effective functions to T-cell to as an the facilitating given overcome acts enabling by IDX attention IDX were death of low-dose little challenges concentration that cell with experimental discovered tumor arrest have cells, Three immunogenic cycle we tumors. tumor induce cell time, can in with first that pathways association the stimulus in for apoptotic properties Therefore, of anticancer potential. range its oncology a on focused via IDX and with studies most Previously, nvitro in α hc srsosbefrteurglto fThle elceoie,sc as such chemokines, cell 1 T-helper of upregulation the for responsible is which fgvna prpit oae (1-2 dosages appropriate at given if , + el oteTE hrb rmtn nitmratvte.While activities. anti-tumor promoting thereby TME, the to cells T 10 α μ ) D a c sa immune-enhancer an as act can IDX M), rdcinb h yn el hsfinding This cell. dying the by production 50 ausrflce t homogenous its reflected values μ )cudsvrl inhibit severely could M) α asdb oso the of loss by caused α + n rv up- drive and -el,were T-cells, + CD8 + Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. ue 1ars yseicls ncci-eedn iae2atvt yp3idpnetidcinof induction p53-independent by activity in- isoflavone, 2 novel kinase a cyclin-dependent Phenoxodiol, in (2005). loss AM specific Senderowicz by & arrest Z, G1 Sheleg MM, duces Facchinetti MF, Aguero References: significance Clinical adds study this What melanoma with patients in immunotherapy to resistance for responsible are cells suppressor melanoma Myeloid-derived with patients in immunotherapy to resistance for responsible are cells suppressor melanoma Myeloid-derived with patients in immunotherapy to resistance for responsible are cells suppressor Myeloid-derived known already is “hot” therapy.What into targeted tumors to “altered” response summary or durable could point “cold” a IDX Bullet a converting that of complement, by chance possible chemotherapy or therefore the is to increasing with, It thereby patients synergize tumors, mechanisms. NPC to resistance sensitize overcome potential or and the therapies, condition provides cancer accumulation IDX of range the of wide growth. inducing behavior tumor of multifaceted abrogate capable the and was Collectively, an sites strategy tumor in such combination better within results whether to this cells our and vital that T confirmed deemed lymphocytes of confirming further are CD8 model, We studies and tumor Further advantage. CD4 animal TME. functional of of the any presence co-expression inside, the confers we the favor potentially co-expression controlling lymphocytes, mechanism could and mechanisms T IDX of, potential the DP by periphery another understand exerted for the observed circumstances at in activation we cells pointed PD1 immune that these effector multiple exhausted results capacity whereas that of migratory our speculate blood, and accumulation such, could profile, from the As cytotoxic tissue render phenotype, lymphoid could memory tissues. secondary IDX peripheral to which to cells through home memory cells central classical CCR7 of memory the into entry was divided CD45RO further facilitate of be would Expression could and which processes. spheroids cells, CCR7 these infiltrate T and to in CD8 involved able memory was were primed marker, CXCL10 of PD-1 that marker and and receptor cells, CD62L tumor of kill expression lower acquiring cells, T • • • • • • • muepiigeeto D ncmiainwt hmteayahee ao oli immune- in goal major achieves chemotherapy destruction with their combination to development in leading drug and IDX oncological “hot”, of into effect micro-tumor “cold” Immune-priming converting by IDX of action Double significance of is interactions tumor-lymphocyte modulates positively IDX abrogate that chemother- and finding tumor The within to accumulation and cancers expansion growth T-cells tumor some enhance cisplatin sensitise with IDX Combining and lines cell cancer in apoptosis apy/radiotherapy causes (IDX) Idronoxil prominent by characterized malignance infiltration. Virus-associated lymphocytic Epstein-Barr an is carcinoma Nasopharyngeal - CD62L - ffco eoyclsbsdo h supinta h rsneo D2 n CCR7 and CD62L of presence the that assumption the on based cells memory effector 11 + Isit uo el.Gvnthe Given cells. tumor into TILs + CD62L nvivo in + eta memory central therapeutic Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. upesmDAidcdSIGmdae yeIINpouto.Cl 159 MJ Cell Herold production. JC, IFN Cambier Natl I RM, J type Lane risk. STING-mediated D, mtDNA-induced cancer Metcalf breast suppress K, and McArthur intake MJ, soy White of Meta-analysis (2006). 98 R Clarke Inst & Cancer L, toxicities. Hilakivi-Clarke treatment BJ, and Trock survival of analysis 67 an Clin 110 W carcinoma: J Oncol Xiao nasopharyngeal Cancer C, Radiother with Zhao CA patients F, 868 2012. Han for F in C, diotherapy Chen subsite Bray S, by AK, Su cancers X, Chaturvedi pharyngeal Agent:Sun R, and Anticancer cavity, Sankaranarayanan an oral A, as lip, Jemal Idronoxil of incidence J, (2020). Ferlay J KD, Targets. Wilkinson Shield Drug & Cancer F, Curr Delebecque Mechanisms. 74 O, and Cell Laczka Activity Mol WD, Fairlie IRF3. and K, MAVS, Porter cGAS, P of Gao Cleavage M, 72 Lv the M, Res via Sha Cancer Production M, D Jing cells. Y, Normolle effector Wang X, T microenvironment AH, Ning cytolytic chemokine Zureikat of the recruitment of HJ, the reprogramming Zeh tumor-selective enhance allows to BF, tissues Junecko tumor anticancer in E, the hyperactivation for Berk target ECTO-NOX cancer. R, (2007). ovarian Muthuswamy DM of Morre 16 & treatment Res C, the Kim Oncol A, for phenoxodiol. Balicki approach isoflavene K, Yagiz novel PJ, a Chueh DJ, Phenoxodiol, Morre (2006). cell 7 T AB Drugs 148 Investig of Alvero Immunol Opin analysis & J Phenotypic Curr HH, age. Fu maturation. dietary of cell G, function of T Mor a Extrathymic study Mol as (1992). profile mice Biochem JP nude urinary Steroid Lake in A J & subsets CW, isoflavonoids. Pierce (1995). new JD, C Kennedy of Nelson metabolism & of M, mode 54 Waring and Biol AY, identification The Reeder kills 94 phenoxodiol, GE, Haematologica phytoestrogens. drug, cells. Kelly anti-cancer T The GE, proliferating (2009). rapidly Joannou DS and Ritchie blasts leukemic & 13 lymphoid MV, Med and Berridge Mol myeloid P, Cell primary Neeson J A JE, Davis Husband vivo. PM, in K, and Herst Ioannou vitro KA, in effects Nicolaou J immunomodulatory M, Int induces isoflavene, Tsiatas anticancer effects. M, antitumour Skopeliti its S, to Georgaki addition D in Brown properties CJ, antiangiogenic Drogemuller 118 potent JJ, Cancer Epi- shows Drew Cancer drug, CN, anticancer carcinoma. Hahn mental P, nasopharyngeal Xia of JR, epidemiology Gamble enigmatic The 15 16 (2006). Prev Oncol Biomarkers HO Lancet demiol Adami meta-analysis. & J MAC-NPC ET, the Ma of Chang WT, Ng update an J, Leclercq carcinoma: S, nasopharyngeal Marguet in 223 A, Pathol Lee J P, Blanchard progression. tumour CD8(+) determine affect microenvironment and sarcoma infiltration Ewing MW T-lymphocyte the Schilham within RM, T Egeler interactions AH, receptor Rutherford Taminiau chemokine-chemokine HJ, D, Baelde 4 SJ, Brown Santos Oncol D, Future A, Berghuis clinic. Leiser to P, bench Rossi from M, phenoxodiol: Kelly AB, Alvero 65 Res Cancer p21WAF1/CIP1. : 167-184. : 2412-2420. : 459-471. : 398-403. : : 1765-1777. 542-548. : 3364-3373. : 299-312. tal. et , tal. et , : 475-482. 12 21) ogtr ucmso nest-ouae ra- intensity-modulated of outcomes Long-term (2014). 21) ppoi apssSprs yeIInterferon I Type Suppress Caspases Apoptotic (2019). : 1620-1629. tal. et , 21) hmteayadradiotherapy and Chemotherapy (2015). tal. et , tal. et , : : : 347-357. 93 e17. 19-31 tal. et , 3735-3743. tal. et , tal. et , 20) hnxdo,a experi- an Phenoxodiol, (2006). 20) nitmratvt of activity Anti-tumor (2008). : tal. et , 1549-1562. tal. et , 21) ppoi caspases Apoptotic (2014). 21) Pro-inflammatory (2011). 20) hnxdo,an Phenoxodiol, (2009). 21) NF-kappaB (2012). 21) h global The (2017). : : 645-655. 51-64. : 3929-3938. : 928-934. Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. fcl rwhi hw.Rslsaerpeettv ftreidpneteprmnsadpeetda the as presented Quantification and experiments (H) independent violet. three crystal of representative with are stained of Results treatment, treatment cis shown. Combinatorial means and/or is (F) growth IDX assay. cell following XTT by of growth Viability analyzed (E) cell was 4 of lines. days to cell images 2, 5 Asterisk respective for 1, in treatment. 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Z, the in Wang H, cancer inhibit Ding prostate synergistically D, of Li risk S, Wu and C, Yao food soy of Meta-analysis from (2005). cells EL 117 fibroblast Spitznagel embryonic & Mouse Biochem L, Cell Yan J (2007). 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XTT by lines cell NPC four in M rCD8 or 21) oamnsrto hnxdo ihdoxorubicin with phenoxodiol Co-administration (2012). 13 + el mn i7 rnyeBadC17 bright CD107a and B granzyme Ki67, among cells T μ rnwl pnIXtreatment, IDX upon transwell m : 392-404. < .5 #p ## 0.05; μ n 2 and M + orsodn to corresponding T < μ IX in (IDX1 M < .0 when 0.005 μ n cis and M .5 **p 0.05; nvitro in Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. D2tetetaoeo ncmiaino i14wsdn.*p?00 hncmae oIXtreatment IDX to compared when p[?]0.05 upon * spheroids 6 done. the Figure was infiltrating Cis1.4 cells pictures of immune 3D combination of of in images Quantification alone. or Representative (H) alone shown. 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Apoptotic using Numbers tumor (F) cytometric (E) points Pre-formed flow software. time ImageJ cocultured. and Fuji indicated were imaging by spheroids the subsequent CellEvent-stained at and and C17/NPC43+ve IC acquired stained of donors the coculture were healthy the determine for images to from used used Representative protocol PBMCs the staining was of was CellEvent the representation and CellEvent (B) Schematic spheroids, (C) outer spheroids with spheroids. the the cis. staining the in and to accumulating IDX of spheroid for constantly layers added LysoTracker values C17 active the was of with metabolically LysoTracker and intensity CellEvent, presumably CellEvent When images of The and Representative uM that concentrations. to 0.1 6000. cis. complementary cis of was InCell and and pattern IDX presence the IDX of using the to concentrations 3 in indicated proportional day cells/well with at 20,000 treated treatments. acquired with then cis were days and and 3 Lysotracker IDX for uM upon 0.2 pre-formed model spheroid were 3D Spheroids a (A) in effects drug of monitoring High-content 5 Figure ANOVA Data 2-way using measured. analyzed p were was ** cells significance boxes. control Statistical p[?]0.05, colored treatment DMSO in * experiments. with highlighted following independent compared are two line cytokines of cytokines representative cell of induced are prominently induction array NPC Most fold Antibody antibodies. indicated relative both. 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Statistical % then collected that flow of such by were E) experiments. data cells, measured Cells cell; Quantitative were dead (F) (effector (%) are days. cells cells PBMCs +. 3 dead FVD+ with the and for contact and cells (E:T) in (FVD) target dye ratio getting line viability cell effector:target before fixable NPC indicated CFSE with of at with plots stained cocultures cytometry T) from flow Representative frequen- cell; the (E) (target of treatments. C17 analysis different cytometry between Flow changes (C) percentage shown. are CD4 patterns of expression cies Differential (blue). 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. rwtotIXa 2hus E uniaino o yoercdt ntepretg fPD of percentage the on data cytometric flow of Quantitation (E) hours. 72 at IDX CD45RO without or and cells) T (memory CD4 vrattlo 1dy fe acrcl nclto n uo ouewscluae.*p?00 and p[?]0.05 * calculated. day was other volume each tumor measured or and were IDX Tumors inoculation mg/kg cell 10 saline. cancer plus received cis after grown mice were mg/kg days had Control which 5 21 tumors mice/group), IDX. mg/kg, of (5 the ***p 20 mg/kg total groups IDX, After 20 6 xenografts a mg/kg plus therefore Tumor into cells. 10 over cis and divided cis, cancer treatment. out mg/kg randomly mg/kg C17 during were dissected 5 5 of volume animals clearly with inoculation Tumor the every-other-day be by (B) mm3, treated not mice 50 shown. could nude approximately not athymic tumor to is in that image established tissue, tumor representative inoculated were organ the its where the group, not cis inside the was from located mouse one deeply For was tumors. cells dissected and T mice increasing Representative (A) with along growth tumor inhibits cis infiltration. and p IDX ** with p[?]0.05, treatment Combinatorial * (DMSO). control to relative 7 IDX Figure of control. presence DMSO the to in compared cells na¨ıvewhen T and exhaustion cells of T analyses memory cytometry Flow (D) hours. 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(2,000 and clustering cells cells heat-map live mononuclear treatment graph-based selected we IDX a data, by upon of followed analysis spheroids ity, unbiased tumor an enable infiltrate To to (A) able are cells T Activated/memory n ehlto rmfu needn ulcmcora aaesGE47,GE31,GE25 and expression tissue. GSE12452 gene normal ENOX2 GSE53819, corresponding of GSE34573, to Analysis datasets compared tumor (H) microarray when Representative patient (G) p[?]0.05 public expression. including * ENOX2. independent ENOX2 type patients of GSE52068. four cell for Some expression from tissues major high patients. methylation with clinical the 11 cells and NPC be malignant from of to contain cells staining found to malignant found histology and were of circled 13 plot were patients. and 15 UMAP tumor 11 * from publicly (F) from harvested a cells lymphocytes. cytometric cells ENOX2. from mainly all live Flow resolution expressing using with total single-cell (D) by enriched at in generated ENOX2 group. clusters plot treatment of UMAP Epithelial cis aforementioned Expression to GSE150430. (E) upon cohort compared cells group. sequencing when available DMSO T to p[?]0.01 + compared CD4 ** CD8 when expressing p[?]0.05 infiltrating group. cells tumor each and on in lymphocytes analysis mouse live one total represents in point cells) (B CD19+ CD8 and (monocyte) CD14+ expressing + + < + .01we oprdt oto ru.()Voi ltsoigtepretg fifitaigcells infiltrating of percentage the showing plot Violin (C) group. control to compared when 0.0001 n CD4 and el hntpspretgs(epcieygtdCD4 gated (respectively percentages phenotypes cells T ) CD8 - CD4 , + /CCR7 + CD8 - CD8 + + + nCD19 in + fCD3 of n CD4 and swl sCD4 as well as ) + - - CD27 nCD4 on ) ouain sltdfo ietmr esrdb o yoer.Each cytometry. flow by measured tumor, mice from isolated populations + CD8 + n¨ v el ust,i h Ncmatet,i h presence the in compartments, IN the in subsets, ) cells (na¨ıve T + + + mn CD3 among CD8 , CD8 , + + 15 ndul-oiieTclssbes ecnae nthe in percentages subsets, cells T double-positive an ndul-oiieTclssbes(epcieygated (respectively subsets cells T double-positive an + ecnae nteI n U compartments, OUT and IN the in percentages ) + CD8 + fCD3 of + nCD45RO on ) + n PD1 and + CD27 < 0.01 + + - , Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. 16 Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. 17 Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. 18 Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. 19 Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. 20 Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. 21 Posted on Authorea 12 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159724553.33561734 — This a preprint and has not been peer reviewed. Data may be preliminary. 22