Leveraging glucan-induced trained immunity for the epigenetic and metabolic rewiring of macrophages to enhance colorectal cancer vaccine response

Domain: neuroscience
Published: 2026-01-28
Editorial outcome: major_revision

Abstract
Colorectalcancer(CRC)remainsrefractorytomostimmunotherapies,withcancervaccinesfailingduetoanimmunosuppressivetumormicroenviron-ment.Here,weshowthatβ-glucan–inducedtrainedimmunityovercomesthesebarriersbyreprogrammingmacrophagesthroughH3K4me3-dependentepigeneticmodificationsandmetabolicrewiring.Infemalemicevaccinatedwithpeptide-coatedadenovirus-basedvaccinePeptiCrad,trainingenhancesglycolysiswithcreatinemetabolismsustainingCXCL9/10production,enablingmacrophagestorecruitNKcellsviaCXCR3.Inturn,NKcellsproduceCCL5,drivingcDC1infiltrationandantigenpresentation,whichtogetheramplifyeffectormemoryCD8⁺Tcellresponses.Moreover,withhumanperipheralbloodmononuclearcellsandCRCpatient-derivedorganoids,trainedmacrophagesboostNKmigration,antigen-specificTcellactivation,andtumorkilling.Thesefindingshighlighttrainedimmunityasapowerfuladjuvanttoreinvigoratecolorectalcancervaccination.

Introduction
Colorectalcancer(CRC)hasattainedsignificantnegativeattentionduetoitbeingthesecond-leadingcauseofcancer-relateddeaths1.More-over, colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide, with an estimated1.9millionnewcasesandalmost1milliondeathsin20202.Despiteitsincidenceplateauingordecreasingamongolderadultsduetothewidespreaduseofscreening,severalstudiesreportedariseinCRC diagnoses among younger adults globally (under 50 years)3. ComparedtolateonsetCRC,youngerpatientshaveahigherproportionofadvancedstageand,despitesignificantlybettersurvivalrates,theoverallsurvival(OS)inmetastaticearlyonsetCRCis18months,highlighting the urgentneed for improved screening strategies andnoveltherapeuticapproaches4.

Traditionaltreatment approaches rely on chemotherapy, radia-tion and radical surgical resection, which, although effective in a subset of patients,are burdened by various side effects and a highrelapserateofaround70-75%,especiallyinmoreadvancedCRCcases4.A shift in the treatment paradigm occurred when cancer immunotherapieswereintroducedintheclinic.Significantadvanceswithimmunecheckpointinhibitors(ICI)wereobservedinthetreatmentofCRC, especially with mismatch repair deficient CRC or high microsatelliteinstabilityowingtohighermutationalburden5–8.Nevertheless,ICIprovedlargelyineffectiveinmismatchrepairproficientormicrosatelliteinstabilitylowCRCwith,accountingfor85%ofallcases9,10.Toovercomethisunmetmedicalneed,variouscancervaccineshavebeendesignedtoenhanceanactiveanti-tumorTcellresponse,andseveralclinical trials have been conducted showing increased overallresponses11–14.Nevertheless,thetumormicroenvironment(TME)wasseenasacommonfactorlimitingtumoreradication.Despitetheformation of an adequate CD8+T cell response/infiltration and IFN-γresponse,thetumor-killingefficacyhasbeendampedbythepresenceof T-regs15–17, myeloid-derived suppressor cells (MDSCs)18–20, tumor-associated macrophages (TAMs)21–24 and immunosuppressivecytokines25–27. Hence, modulation of the TME could be an effective strategytoenhancecancervaccinesinCRC.

TrainedimmunitywasdescribedbyNeteaandcolleaguesasanaspecificmemoryresponseoftheinnateimmunesystem28.Itinvolvesan epigenetic and metabolomic reprogramming of innate immunecells, when exposed to specific stimuli, resulting in an enhancedresponsewhenre-exposedtoaheterologousstimulus.Manystudieshaveshownaprotectionagainstaheterologouschallengeofpathogensincludingbacteria29–31,viruses32–36orfungi.Withrespecttocancer,only recently hasthere been studies implicating tumor cells asstimuli for trained immunity. These effects have been seen inbreast37,38,pancreatic39,40andlungcancerwhereanoverallanti-tumorinflammation was noticed in all studies. Specifically, a reduction ofMDSCandreprogrammingofTAMandmonocytestowardsanti-tumorhavebeenobservedleadingtoanincreaseinICItherapyresponse39,40.Therefore,itisbelievedthattrainedimmunitycouldenhancecancervaccination by modulating the TME, specificallythe innate immunepopulations.Moreover,italsobelievedthattrainedimmunitycouldfurtherboostthestrengthofelicitedadaptiveimmuneresponsesbystrengtheningtheinnateimmuneresponse.Thisisaconceptthathasbeenhypothesizedbutneverstudied.

In this work, we utilize beta-glucan as an inducer of trainedimmunityandtestwhetheritcouldenhancetheeffectofPeptiCrad(PC)41,42,acancervaccineplatforminwhichtumor-specificpeptidesareelectrostaticallycoatedontothesurfaceofadenoviruses,therebycombiningefficientviraldeliverywithantigen-specificTcellpriming.PCiscurrentlyunderclinicalevaluation(NCT05492682).WhentrainedimmunityisinducedinCRC-bearingmice,vaccinationisshowntobeenhanced.MetabolomicstudiesrevealarewiringofTAMscausinganincreaseintheWarburgeffectandepigeneticchanges.Single-cellRNAsequencing reveals that such TAMs upregulate CXCL9 and CXCL10leadingtoanincreaseinNKcellinfiltration.ThisincreaseofNKcellsresultsintheexcretionofCCL5inducingconventionaldendriticcells1(cDC1) populations further enhancing T-cell responses. In humanstudies,wealsoshowthatPBMCsinducedbytrainedimmunityhavehigher CD8+T cells responses when treated with a cancer vaccineleading to an enhanced tumor killing in CRC-patient derivedorganoids.

Results
Glucan-inducedtrainedimmunityenhancesvaccineresponse
To explore the immunological effects of glucan-induced trainedimmunity, we re-analysed single-cell RNA sequencing data from apreviouslypublishedstudy40.InthatstudyC57BL/6miceweretreatedwitheitherPBSorwholeglucanparticlesderivedfromcellwallsof yeast(WPG), and pancreas tissuewascollected atdays3 and 7post-treatment. Distinct shifts in immune cell populations wereobservedbetweenPBS-andWPG-treatedmice(Fig.1A,SupplementaryFig.1A).Notably,clusters7,11,and12increasedprogressivelyinWPG-treatedmice(Fig.1B)andwereidentifiedasChil3+Ly6Cmacrophages and monocytes, suggesting their involvement in trainedimmunity.

Averagegeneexpressionrevealedincreasedantigenprocessingand presentation genes in WPG mice at both time points (SupplementaryFig. 1B–C).Hif1α,a hallmark of trained immunity, was also
upregulated(SupplementaryFig.1D).DEGanalysisofclusters7,11,and12confirmedupregulationofantigenpresentationgenes(Fig.1C).Incluster11,b2m,h2-k1,ctss,andh2-q7wereelevatedatday7(Fig.1D–E),whilecluster7showedincreasedctsc,h2-dma,h2-ab1,andh2-aa,andcluster12upregulatedb2m.ThesechangesalignwithenhancedMHC-IandMHC-IIpathways,supportingadaptiveimmunepriming.Furthermore, cxcl9 and cxcl10 were upregulated (Supplementary Fig. 1E),suggesting increased NK and T cell recruitment. Importantly, thisfocused analysis of clusters 7, 11, and 12—emphasizing antigen presentationand NK/Tcellattraction—was notexplored in the originalGellerdataset.

Sincethisanalysiswasrestrictedtothepancreasandimmunecellpopulationsmaydiffer,wefurtherinvestigatedwhetherWPG-inducedtrained immunity could enhance a vaccine response systemically.C57BL/6micewerepre-treatedwithPBSorWPGandvaccinatedwithadenovirus-basedPCcarryingSIINFEKL(Fig.1F)asaproofofconcept.FlowcytometryrevealedthatSIINFEKLstimulationinducedsignificantshiftsinTcellmemorysubsets(SupplementaryFig.2A,andSupplementaryFig.3A).Trainedmiceshowedreducedcentralmemory(CM)(CD44+CD62L+) and increased effector memory (EM) (CD44+CD62L–) CD8+T cells (Fig. 1G), a phenotype linked to robustanti-tumor responses. Moreover, CD8+T cells from trained mice expressedhigherIFN-γandTNF-α(Fig.1H–I),withincreasedcytokineproductionpercell(SupplementaryFig.3B).IL-2wasnotelevated(SupplementaryFig.3C).Additionally,SIINFEKLstimulationinducedIL-2,TNF-α,andIFN-γinCD4+Tcells(SupplementaryFig.3D),likelyreflectingindirectCD8+Tcelleffects43,44.Together,theseresultsdemonstratethatWPG-inducedtrained immunity enhances antigen presentation in myeloidcells and promotes effector memory CD8+T cell responses,strengtheningvaccine-inducedimmunity.

ImmunityenhancesvaccineresponseinbothaprophylacticandtherapysettingsinCT26bearingmice
After observing the effects of our initial in vivo vaccine model, weproceededtotestourapproachinacoloncancersettingusingtheCT26model.Tomimicaprophylacticsetting,Balb/cmicewerefirsttreatedwitheitherPBSorWPG(Fig.2A)beforetumorimplantation.Following this, we utilized the same adenovirus-based vaccine (PC),thistimecoatedwithavalidatedimmunogenicCT26-derived/specificpeptide(Sylpeptide)44.Asshown,micetreatedwiththecombinationofWPGandPeptiCRAd(Trained+PC)exhibitedthestrongesttumorcontrol,PCaloneshowedmoderatesuppression,andWPGalonehadnoeffectcomparedtomock(Fig.2B,SupplementaryFig.4A).Thus,trained immunity alone was insufficient, but it primed the immunesystemtoenhancevaccineefficacy.

Aftersacrificingthemice,weanalyzeddendriticcellsubsetsindraininglymphnodes(Fig.2C,SupplementaryFig.5A).WhilecDC2(CD103- MHCII+ CD11c+) cells dominated across groups, WPG andTrained+PCtreatmentincreasedcDC1(CD103+MHCII+CD11c+)frequency,withTrained+PCshowingthestrongesteffect.

Next,wenextassessedthesystemicimmuneresponsebyisolatingsplenocytesfromtreatedmiceandstimulatingthemexvivowith
either PBS, adenovirus, or the Syl peptide. With PBS stimulation,Trained+PCmiceshowedelevatedEMTcellsinbothCD8+(CD44+CD62L-)(Fig. 2D) and CD4+(Fig. 2E)compartments, indicating systemic priming. Upon Syl peptide restimulation, Trained+PC splenocytesagainexhibitedmoreEMTcellsandfewerCMTcells(CD44-CD62L-) in CD8+ (Fig. 2F) and CD4+ (Fig. 2G) compartments, confirming peptide-specific memory. In contrast, WPG alone failed toinduceSyl-specificresponses,thoughsplenocytesrespondedrobustlytoadenovirus, consistent with trained immunity boosting antiviralpathways.

To assess therapeutic potential, we tested established CT26tumors.WPGwasadministeredpost-implantation,followedbyvaccination(Fig.2K).Again,Trained+PCmiceexhibitedthelowesttumorburden(Fig.2L–M),whereasMock,PC,orWPGalonehadlittleeffect.TMEanalysisshowedincreasedmonocyte(Fig.2N)andTAMinfiltration (Fig. 2O) in Trained and Trained+PC groups, while neutrophilsremainedunchanged(SupplementaryFig.6A).TAMsexpressedhigherCD80(SupplementaryFig.6B)andmonocyteshigherCD86(SupplementaryFig.6C),indicatingactivation.

Together,theseresultsdemonstratethatWPG-inducedtrainedimmunity enhances CRC vaccination in both prophylactic andtherapeutic contexts. This synergy involves expansion of cDC1,systemiceffectormemoryTcellpriming,increasedcytokineproduction, and remodeling of the TME to favor monocyte/TAMactivation.

MetabolomicprofilingofCD11b+cellsrevealsmetabolicreprogramming
Trainedimmunityhasbeenpreviouslyshowntoalterthemetabolicfunctionofmyeloidcells.Tofurtherinvestigatethemetabolicstateoftumor-infiltratingCD11b⁺cells,weperformedacomprehensivemetabolomicanalysisfollowingtreatment.GiventhatCD11b⁺cellsaretheprimaryimmune populationaffected by our strategy, we sought todeterminehowtheirmetaboliclandscapechangesuponexposuretoPC versus WPG combined with PC (Trained+PC). Tumors were harvested,CD11b+cellswereFACS-sorted,andsubjectedtometabolomicprofiling(Fig.3A,SupplementaryFig.7A).

Principal component analysis(PCA) revealed distinct clusteringbetweenthetwogroups,withPC1andPC2accountingfor62.2%and24.5% of the variance, respectively (Fig. 3B), highlighting a cleardivergenceinmetabolitecompositionafterTrained+PC.Comparativeanalysisrevealedpronouncedincreasesinmultipleaminoacids(alanine, serine, glutamic acid, valine, asparagine, malic acid, arginine),fattyacids,andkeyintermediatesofthecreatinepathway(creatine,creatine phosphate, creatinine) (Fig. 3C, and SupplementaryFig.7B–C).Differentialmetaboliteanalysisconfirmedtheupregulationof several amino acids and creatinine (Fig. 3D, and SupplementaryFig. 7D). Pathway impact analysis further identified upregulation ofaminoacidbiosynthesis(notablyarginine,serine,glutamicacid),fattyacid synthesis, and pathways linked to aerobic glycolysis and theWarburgeffect,supportedbyelevatedlactate(Fig.3E).Metabolitesetenrichmentanalysisreinforcedthesefindings,showingenrichmentofglycolytic,aminoacid,andlipidpathways(Fig.3F).Collectively,thesedata suggest Trained+PC promotes a coordinated shift toward biosyntheticand energy-generatingprograms.Giventhesefindings,wesought to explore how different metabolic pathways were interconnected (Supplementary Fig. 7D). Correlation analysis revealedstrong associations between nucleotide biosynthesis, amino acidmetabolism,fattyacidsynthesis,andtheWarburgeffect.Tovisualizetherelationshipsamongthesemetabolicpathways,weconstructeda network-based metabolite interaction map (Supplementary Fig. 7E).This analysis revealed a clear interconnection between amino acidmetabolism,fattyacidbiosynthesis,andnucleotidesynthesis,furthersupportingourhypothesisthatTrained+PCtreatmentinducesacoordinated metabolic shift towards biosynthetic and energy-generatingpathways.

To functionally validate these observations, weperformed Seahorseextracellularfluxassaysonisolatedmurinemacrophages,duetotheirhighabundanceinthetumor(Fig.2J).Following18hofexvivostimulation with PBS or WPG, trained macrophages exhibitedsignificantlyhigheroxygenconsumptionrates(OCR)acrosstwoplatingdensities (40k and 80k cells) (Fig. 3H), with increases in basal andmaximalrespiration(SupplementaryFig.8A–C).Notably,amajorpartofthebasalOCRwasattributabletonon-mitochondrialoxygenconsumption(SupplementaryFig.8D).Inparallel,trainedmacrophagesdisplayedelevatedextracellularacidificationrates(ECAR),withhigherlevelsbothatbasalconditionsandafterinhibitionofthemitochondrialATPsynthesis,consistentwithanaugmentedglycolyticactivity(Fig.3I)andbasalglycolysislevels(SupplementaryFig.8D).

Toprobethekineticsofthismetabolicrewiring,werepeatedtheassay after only 2h of stimulation. Under these conditions, OCRremained comparable between groups (Fig. 3K; and SupplementaryFig. 8F–H). Again, most OCR reflected non-mitochondrial oxygenconsumption (Supplementary Fig. 8I). Yet, ECAR was already significantlyincreasedintrainedmacrophages(Fig.3L),accompaniedbyelevated basal glycolysis (Supplementary Fig. 8J). These resultsdemonstratethatWPGrapidlyreprogramsmacrophagestowardglycolysis,consistentwithaWarburg-likephenotypethatisestablishedwithinhoursandfurtherreinforcedovertime

Together, metabolomic and bioenergetic profiling converge toshow that trained macrophages undergo broad metabolic rewiringcharacterizedbyenhancedaminoacidandlipidbiosynthesis,creatinemetabolism,andapronouncedshifttowardaerobicglycolysis.Thisreprogramming provides the energy and biosynthetic capacityrequiredtosustainpro-inflammatoryandanti-tumorfunctionsinthecontextofvaccination.

Single-cellRNAsequencingrevealsadistinctinnateimmunelandscapefollowingtrained+PCtherapy
ToassesshowTrained+PCalterstheinnateimmunelandscapeintumors, we performed scRNA-seq on sorted CD11b+ cells fromtumor-bearingmicetreatedwithPCorTrained+PC.Thisallowedusto capture treatment-driven changesacross the myeloid compartment. Clustering revealed diverse innate populations, with macrophages,monocytes,andNKcellsasdominantsubsets(Fig.4A,andSupplementaryFigure9A).Clusters0,3,2,7,10,and1(macrophages, monocytes, NK cells) were significantly expanded inTrained+PC, demonstrating remodeling of the TME (Fig. 4B, C).Consistent with this, Trained+PC upregulated cytokines such ascxcl9 and cxcl2 (Supplementary Fig. 9B), suggesting enhancedrecruitmentofTandNKcells.Analysisdidrevealaclearexpressionofitgam(CD11b)onmajorityofclusters(SupplementaryFig.9C),with minimal contamination, demonstrating successfulcompensatoryenrichment.

Wenextsoughttoidentifythefunctionalprogramswithintheseclusters. Stratification revealed two major inflammatory patterns(Fig.4D).First,clusters2(chil3+ly6Chimacrophages,SupplementaryFig.9D),14(NK),13(cDC1),7(monocytes),and9(ILCs)upregulatedfcgr1,ccl9,cxcl9/10,cd86,andirf5,consistentwithapro-inflammatoryphenotype.Expressionoflyz2andtnfaip2intheseclusters(SupplementaryFig.9E)furthersupportedmyeloid-driveninflammation,withcluster2showingthestrongestresponse.Second,clusters1(NK),8(monocytes),and11(NKT)displayedatypeIIFNsignature(Ifngr1).Wealso examined β-glucan sensing and found clec7a (dectin-1), a keyreceptor involved in β-glucan recognition41,42, broadly expressed ininflammatoryclusters(SupplementaryFig.9F),exceptNK/NKTcells,suggestingindirectNKactivationviacytokinesandchemokinecrosstalkwithinthetumormicroenvironment.TotestwhetherTrained+PCshifted cells toward suppression, we analyzed anti-inflammatorygenes. Few were detected (Fig. 4E): cluster 2 macrophages showedlimitednos2/arg1expression,butthiswasconfinedtoaminority.Thus,Trained+PC promoted durable inflammatory activation rather thancompensatorysuppression.

Tofurtherdissectamechanisnofaction,weperformedajoint-pathway enrichment analysis, integrating scRNA-seq data with previouslyobtainedmetabolomicscreening(Fig.4F).Notably,pathwaysassociatedwithTcellactivation,includingTCRsignalingandTh1/Th2differentiation, were significantly enriched, suggesting that Trained+PCtherapyenhancedadaptiveimmunepriming.Additionally,chemokinesignalingandJAK-STATpathwaysemergedaskeyregulatorynetworks,indicatingthatcytokine-drivenimmunemodulationwasacentral feature of the treatment-induced changes. Interestingly, NKcell-mediatedcytotoxicitywasamongthemostsignificantlyupregulatedpathways,despiteNKcellsthemselvesnotexpressingdectin-1.This strongly suggests that their expansion and activation weresecondary to inflammatory cues from other myeloid populationsratherthandirectWPGsignaling.

TodissectthemechanismsdrivingNKcellexpansion,weanalyzedmonocyte and macrophage clusters, asthese cells areprimary producersofcytokinesknowntorecruitandactivateNKcells(Fig.4G).Cluster2macrophagesexhibitedamarkedlyheightenedinflammatoryprofilecomparedtocluster7monocytes,suggestingadominantroleinorchestratingtheimmuneresponse.Expressionanalysisconfirmedthat cluster 2 macrophages strongly upregulated cxcl9, cxcl10, andil15—keyfactorsknowntodriveNKcellrecruitment,proliferation,andcytotoxicity (Fig. 4H). These findings imply that trained immunity-induced macrophages (Chil3+ Ly6Chi macrophages) act as centralregulators of NK cell expansion and function within the tumormicroenvironment.

GiventhesubstantialNKcellinfiltrationobservedinTrained+PC-treatedtumors,wefurthercharacterizedcluster1NKcellstodefine theirfunctionalphenotype(Fig.4I),basedonpreviousclassificationmethods45.Byclassifyingthesecellsintoresting,cytokine-responsive,activated, and type I IFN-responsive subpopulations, weobserved apredominantenrichmentofcytokine-responsiveNKcells(Fig.4I).Thiswasaccompaniedbyrobustupregulationofchemokinessuchasccl5,ccl4,andxcl1(Fig.4J),whichareknowntofacilitatetherecruitmentofconventionaldendriticcells(cDCs).Moreover,amongthetopgeneswith the principal component analysis was ccl5 (SupplementaryFig.9G)furtherdemonstratingitsimportance.Hence,thechemokine-producingNKphenotypesuggestsacriticalroleforNKcellsinshaping subsequentantigen-presentingcell(APC)activation.Moreover,cluster1 NK cells from Trained+PC treated mice were seen to have similarlevelsofpdcd1,tigit,havcr2indicatingnoexhaustioneffects(SupplementaryFig.10A).ThiswasfurtherconfirmedwhenanalysingtheNKcellsfromthetumormicroenvironmentofmicetrainedaftertumorimplantation(SupplementaryFig.10B).

ToexaminethedownstreamimpactonAPCfunction,weanalyzedclusters12and13,representing plasmacytoid dendritic cells(pDCs)andconventionaldendriticcells(cDC1),respectively(Fig.4K).cDC1s(cluster 13) showed marked upregulation of antigen-presentationgenescd80,cd83,andh2-Aa(Fig.4K,L),consistentwithanactivatedphenotype. Thus, Trained+PC did not merely expand cDC1s butenhancedtheirfunctionalcapacity.

Together,theseexperimentsrevealapossiblesequentialactivation cascade: trained macrophages (cluster 2) secrete Cxcl9/10 andIl15, recruiting NK cells; NK cells secrete Ccl5/Xcl1, enhancing cDC1infiltration and activation; cDC1s then prime T cells. Thismacrophage–NK–cDC1axiscouldprovideamechanisticexplanationforthesuperiorefficacyofTrained+PCcomparedtoPCalone.

CreatineRegulationinTrainedImmunityEnhancesPro-InflammatoryCytokineReleaseandNKcellinfiltration
Todissectthemetabolicrequirementsoftrainedimmunity,weenriched splenicCD11b⁺ cells, exposed them to PBS or WPG, and restimulatedthemwithadenovirusinthepresenceorabsenceofmetabolicinhibitorstargetingglycolysis/mTOR(rapamycin),HIF-1αstabilization(ascorbate), or creatine uptake (β-GPA) (Fig. 5A). PBS-treated cellsproducednoTNF-α,whereasWPG-trainedcellsshowedrobustTNF-αandIL-6induction,bothofwhichweresignificantlyreducedbyinhibitors(Fig.5A,B).SinceourscRNA-seqdatahighlightedCXCL9andCXCL10 as key chemokines strongly upregulated in trained macrophages,wenextassessedtheirsecretion.Consistentwithoursingle-cellfindings,trainedCD11b⁺cellssecretedsignificantlyhigherlevelsofCXCL9 and CXCL10 compared to PBS controls. However, secretionwas abolished when rapamycin, ascorbate, or β-GPA were added,demonstratingthatbothglycolyticandcreatine-dependentmetabolism support the pro-inflammatory reprogramming of trainedmacrophages.

Sincetheinitialexperimentstestedinhibitoreffectsonlyduringsecondary stimuation, we repeated the assays by adding inhibitorsduringtheprimingstageofWPGtraining.Asexpected,trainedcellsproducedelevatedIL-6,TNF-α,CXCL9,andCXCL10uponadenovirusstimulation,yetthiswasseverelybluntedbyallinhibitorswhengivenatpriming(SupplementaryFig.11A–E).Moreover,whenwewashedβ-GPAandsupplementedwithcreatine,CXCL9andCXCL10expressionswererescued(SupplementaryFig.11DandE).Todirectlytestwhethercreatinemetabolismimpactsepigeneticprogrammingoronlyeffectorfunction,wefocusedonmacrophages,astheyemergedinoursingle-cellanalysisasthekeytrainedpopulationdrivingtheNK–cDC1axis.CD11b⁺F4/80⁺macrophageswerethereforeenrichedandstainedforH3K4me3, a histone mark associated with trained immunity. ComparedtoPBScontrols,WPG-trainedmacrophagesshowedamarkedincrease in H3K4me3 after adenovirus stimulation (SupplementaryFig.11F).Thisenrichmentwaspreservedeveninthepresenceofβ-GPA(SupplementaryFig.11G),indicatingthatcreatinedoesnotinfluencetheacquisitionoftrainedepigeneticmarksbutrathersustainseffectorfunctionduringsecondarychallenge.WenextassessedwhetherWPG-induced epigenetic remodeling regulates expression of the creatinetransporterSlc6a8.ChIP-qPCRforH3K4me3showednoenrichmentattheSlc6a8locus(SupplementaryFig.11H).However,RT-qPCRrevealedasignificantincreaseinSlc6a8transcriptlevelsintrainedmacrophagescomparedtoPBS-treatedcontrols(SupplementaryFig.11I),suggestingthat upregulation of creatine transport occurs independently ofH3K4me3-mediatedepigeneticinduction.

ToassesstheroleofcreatineinadaptiveimmunityinducedbyPC,we tested whether β-GPA treatment affected tumor control. CT26-bearingmiceweretreatedwithPBSorWPG(trained)andvaccinatedwithPC,withasubsetreceivingdailyβ-GPAtoblockcreatineuptake(Fig. 5C). As expected, Trained+PC provided the strongest tumorcontrol (Fig. 5D, and Supplementary Fig. 12A). In contrast, β-GPAtreatment partially impaired thiseffect,with tumors growing largerthan in Trained+PC but remaining smaller than in mock controls(Fig.5C,D,andSupplementaryFig.12A).β-GPAalonedidnotaffecttumorgrowth,confirmingithasnointrinsicanti-orpro-tumoractivity.

Todissectimmunealterations,weanalyzedtumorinfiltrationofmacrophages and NK cells (Supplementary Fig. 11B). Macrophagenumbers were unchanged between Trained+PC and Trained+PC+β-GPAgroups(Fig.5EandF),whereasNKcellinfiltrationwasmarkedlyreducedinβ-GPA–treatedtumors(FiguresGandH).Thisalignswithourearlierfindingthatβ-GPAsuppressesCXCL9/CXCL10secretionfromtrainedCD11b⁺cells,chemokinescriticalforNKrecruitment.

To evaluate epigenetic remodeling in vivo, we stained tumor-derivedCD11b⁺F4/80⁺macrophages.Trained+PCincreasedH3K4me3compared to PBS controls (Fig. 5I, J). Importantly, this enrichmentpersisteddespiteβ-GPA,reinforcingthatcreatinedoesnotdriveepigeneticprogrammingbutinsteadsupportseffectorresponses.Together,theseresultsdemonstratethatcreatinemetabolismisacentraldriver of the effectorphase oftrained immunity in CRC, sustainingcytokineandchemokineproduction(includingCXCL9andCXCL10)and enabling NK cell recruitment, without altering the underlyingepigeneticreprogramming.

Macrophage-NKcellcrosstalkpromotesDCinfiltrationandCD8+Tcellmemoryintrainedimmunity
ToclarifythefunctionalrolesofmacrophagesandNKcellsintrainedimmunity–mediated tumor control, we selectively depleted thesesubsetsinCT26-bearingmicepriortoWPGtrainingandPCvaccination(Fig.6A).Flowcytometryconfirmedefficientdepletion(Supplementary Fig. 13A). Tumor growth showed that loss of either subsetimpaired control, while dual depletion abolished efficacy (Fig. 6B,Supplementary Fig. 13B), demonstrating non-redundant, complementaryroles.

We next asked how these cells influence systemic immunity.SplenocytesfromTrained+PCmicecontainedabundantEMCD8+Tand CM CD8+T cells after Syl peptide restimulation (Fig. 6C),whereasdepletionofmacrophagesorNKcellsreducedbothpopulations (Fig. 6D). This correlated with impaired DC recruitment,particularly cDC1, as shown by flow cytometry (SupplementaryFig. 14A, B). Functionally, CD8⁺ T cell cytokine production (IFN-γ,TNF-α) was strongly reduced in depleted groups (SupplementaryFig.14C,D).Together,theseresultsindicatedthatmacrophagesandNKcellstogetherformacriticalupstreamaxisnecessarytoestablisheffectiveadaptiveimmunity.TofurtherdefinetheroleofTcells,werepeatedtheexperimentsinCT26-bearingmicetreatedwithTrained+PC but depleted of CD8⁺ T cells (Supplementary Fig. 15A). Successfuldepletionwasconfirmedbybloodanalysis(SupplementaryFig. 15B), and we observed that these mice completely lost tumorcontrol(SupplementaryFig.15C).Thus,trainedmacrophagesorNKcellsaloneareinsufficienttomediatetumorcontrolintheabsenceofCD8⁺Tcells.

To dissect this relationship further, we performed adoptivetransferexperimentswithPBS-orWPG-treatedmacrophagesandNKcellsintoCT26-bearingmicethatweresubsequentlyvaccinatedwithPC(Fig. 6E, Supplementary Fig. 16A and B). Remarkably, only micereceivingtrainedmacrophagesincombinationwithPCexhibitedsignificanttumorgrowthreduction(Fig.6F).TransferoftrainedNKcellsalonedidnotaffecttumorburden,establishingmacrophagesastheprimary initiating population in this circuit. Consistently, trainedmacrophage transfer led to higher frequencies of splenic EMCD8+Tcells(Fig.6G)andenhancedcytokineresponsesuponantigenrestimulation,withsignificantlyelevatedIFN-γandTNF-αproductioninCD8+Tcells(Fig.6H,I).

Analysisofthetumormicroenvironmentfurthersupportedthishierarchy. Mice receiving trained macrophages plus PC displayedincreasedintratumoralmacrophages(SupplementaryFig.17A)andNKcells(SupplementaryFig.17B).Importantly,theseNKcellswerefunctionallyactivated,asdemonstratedbyincreasedperforinexpression(Supplementary Fig. 17C). While no changes were detected in plasmacytoidDCs(pDCs;SupplementaryFig.17D)orcDC2populations(Supplementary Fig. 17E–F), cDC1 tumor infiltration was markedlyincreased in the trained macrophage + PC group (SupplementaryFig. 17G). This increase coincided with the expansion of EM CD8+ T cells in the TME (Supplementary Fig. 17H), reinforcing the linkbetween macrophage-driven NK cell recruitment, subsequent cDC1activation,andeffectiveTcellpriming.

To directly assess the crosstalk between macrophages and NKcells,weemployedatranswellmigrationassayinwhichCFSE-labeledNKcellswereplacedintheupperchamberandmacrophagesisolatedfromTrained+PCormocktumorswereplacedbelow.NKcellsshowedgreater migration toward Trained+PC-derived macrophages comparedto controls (Fig. 6J, K). Microscopy confirmed this enhancedrecruitment. Importantly, pre-treatment of NK cells with a CXCR3-blocking antibody markedly reduced migration, demonstrating thatmacrophagesrecruitNKcellsviatheCXCL9/CXCL10–CXCR3axis.

WenextaskedwhetherNKcellscould,inturn,recruitdendriticcells(DCs).Usingthesametranswellsystem,NKcellsisolatedfromtheTME of Trained+PC-treated mice, but not from mock tumors, promotedDCmigration(Fig.6L,M).ThiseffectwasabrogatedbyCCL5blockade, implicating NK cell–derived CCL5 in DC recruitment(Fig. 6L). FLT3L blockade did not affect DC recruitment, furtherhighlighting the need of CCL5 (Supplementary Fig. 18A). Notably,mockNKcellstreatedwithWPGinvitrodidnotincreaseDCmigration,suggestingthatNKcellfunctionalactivationrequiressignalspresentwithinthetrainedTME rather than direct β-glucan exposure.

To investigate the transcriptional basis of these observations, weperformed ChIP-qPCR on macrophages isolated from PBS- or WPG-treated mice. Trained macrophages displayed elevated H3K4me3enrichment at cxcl9 and cxcl10 loci (Supplementary Fig. 18B), consistentwithepigeneticrewiringdrivingsustainedchemokineproduction.Incontrast,NKcellsfromeitherPBS-orWPG-treatedmicedidnotshowH3K4me3enrichmentatccl5,ccl4,orflt3loci(SupplementaryFig.18C).Similarly,NKcellsisolatedfromtheTMEofTrained+PCmice lackeddetectableepigeneticmarksatthesegenes.However,RT-qPCRofwhole-celllysatesrevealedthatNKcellsfromTrained+PCtumorsdidexpressincreasedlevelsofccl5mRNA(SupplementaryFig.18D).TheseresultssuggestthatNKcellsarenotepigeneticallyrewiredbytrainedimmunitybutareinsteadsecondarilyactivatedwithintheTME,likelythroughmacrophage-derivedcues.

BecausecreatinemetabolismsustainedmacrophageCXCL9/10output,weaskedwhetheritalsomaintainsthemacrophage–NK–cDC1axis. Adoptive transfer of trained macrophages plus PC vaccinationwascombinedwithβ-GPAtreatment(SupplementaryFig.19A).β-GPAimpaired tumor control (Supplementary Fig. 19B) and reduced NKinfiltration (Supplementary Fig. 19D) without affecting macrophagenumbers(SupplementaryFig.19C).

Todeterminewhethercreatinemetabolismitselfwasepigeneticallyimprinted,weperformedChIP-qPCRforH3K4me3atthecreatinetransportergeneslc6a8.Noenrichmentwasdetectedintrainedmacrophages (Supplementary Fig. 119E), yet RT-qPCR of cell lysatesrevealedincreasedslc6a8expression(SupplementaryFig.19F).Thus,trained macrophages acquire enhanced creatine reliance, but thismetabolicadaptationistranscriptionalratherthanepigenetic.

Together,theseexperimentsestablishthattrainedmacrophagesaretheinitiatingpopulation,epigeneticallyrewiredtosecreteCXCL9/10,whichrecruitNKcellsviaCXCR3.NKcellsthenamplifyimmunityby producing CCL5, driving cDC1 infiltration and T cell priming.Creatine metabolism sustains this axis by maintaining macrophagechemokine output, highlighting a hierarchical, metabolically supported macrophage–NK–cDC1–T cell circuit critical for anti-tumorefficacy.

TrainedimmunityenhancesvaccineefficacyintheMC38andcolon-26tumormodel
To determine whether the effects of trained immunity extendedbeyondtheCT26model,wenexttestedtheMC38colorectalcancermodel.Miceweredepletedofmacrophagesand/orNKcellsbeforePBSorWPGtrainingandpeptidevaccination(Fig.7A).Trained+PCtreatment produced superior tumor control, with many mice achievingcompleteclearance(Fig.7B,andSupplementaryFig.20A).Depletionofeithersubsetpartiallyimpairedprotection,whilecombineddepletionabolishedit,mirroringCT26results.

Wethenevaluatedadaptiveimmunity.Splenicprofilingshowedmarked expansion of EM and CM CD8⁺ T cells in Trained+PC mice(Fig.7C,D,andSupplementaryFig.20B),togetherwithelevatedIFN-γand TNF-α after peptide stimulation (Fig. 7E, F, and SupplementaryFig. 20C, D). These responses were lost with macrophage or NKdepletion. In draining lymph nodes, Trained+PC mice displayedincreasedcDC1frequencies, consistent with enhanced cross-presentation,whereasdepletionshiftedthebalancetowardcDC2dominance(Fig.7G,andSupplementaryFig.20E).

Becausecreatinemetabolismsustainedchemokineproductioninearlierexperiments,weaskedwhetheritalsoregulatedTrained+PCefficacyinvivo.Micereceivedβ-GPAtoblockcreatineuptake(Fig.7H).While Trained+PC again provided robust control, β-GPA treatmentpartially reversed protection (Fig. 7I, and Supplementary Fig. 20F),reducingEM/CMCD8⁺Tcells(Fig.7J),IFN-γandTNF-αproduction(Fig.7K,L),andcDC1accumulationindraininglymphnodes(Fig.7N).TheseresultsconfirmthatcreatinemetabolismisrequiredforNK-andDC-dependentamplificationoftrainedimmunity.

Finally, to validate findings in a more immunosuppressive context,weusedtheColon-26model,whichisAPC-mutant,refractorytoPD-1/PD-L1therapy,andconsideredmoreclinicallyrelevanttohumanCRC (Supplementary Fig. 21A). Vaccination alone failed to controltumors, whereas Trained+PC significantly suppressed growth(Supplementary Fig. 21B, C). Trained+PC tumors showed increasedmacrophage infiltration (Supplementary Fig. 21D), greater NK accumulation(SupplementaryFig.21E),andenhancedcDC1recruitmentindraining lymph nodes (Supplementary Fig. 21F). Splenocytes fromTrained+PCmiceproducedhigherIFN-γandIL-2uponA1H5peptiderestimulation(SupplementaryFig.21H–I)andexhibitedincreasedEMCD8⁺Tcells(SupplementaryFig.21J).

Together, these results demonstrate that the macrophage–NK–cDC1axisdrivesvaccineefficacynotonlyinCT26butalsoinMC38andthehighlyimmunosuppressiveColon-26model.Trained immunity consistently promoted cDC1 recruitment, robusteffector and memory CD8⁺ T cell responses, and dependence oncreatinemetabolism,supportingitspotentialasabroadlyapplicableenhancerofCRCvaccination.

TrainedImmunityEnhancesVaccineResponsesandTumorKillinginCRCPatientDerivedOrganoids
Toextendourpreclinicalfindingstohumansettings,wefirstexaminedtheclinicalsignificanceofmacrophagesubsetsusingpubliclyavailabledatasets.Inacohortof163patientswithCRC(cBioPortal),stratificationbyCD206expression—amarkertypicallyassociatedwithM2-likemacrophages—revealedthatpatientswithhighCD206expressionhadsignificantlylongerdisease-freesurvivalcomparedtothosewithlowexpression(Fig.8A).FurtherstratificationoftheCD206-highcohort(n=123) based on CXCL9expressiondemonstrated thatindividuals with elevated CXCL9 levels displayed superior outcomes (Fig. 8B).TheseresultssuggestthatmacrophagesacquiringaCXCL9⁺phenotypemaypromoteimprovedanti-tumorimmunity,consistentwiththemacrophage–NK–cDC1axisweidentifiedinourmurinemodels.

We next sought to determine whether WPG-induced trainedimmunitycouldberecapitulatedinhumanimmunecellsandwhetherit could augment vaccine responses. PBMCs and monocytes fromhealthydonorsweretrainedwithWPGfor24h,washed,andsubsequentlystimulatedwithadenovirus,theadjuvantusedinthePCvaccine.Whilenon-trainedmonocytesproducedmodestIL-6andTNF-αresponses, trained monocytes exhibited markedly higher cytokinefrequencies and concentrations following adenovirus stimulation(SupplementaryFig.22A).Differentiationoftrainedmonocytesintomacrophagesordendriticcellsretainedtheseeffects:trainedmacrophages produced significantly higher IL-6 and TNF-α levels, whiletrainedDCsexhibitedincreasedIFN-γsecretionandelevatedexpression of CD83, a costimulatory molecule critical for T cell priming(Supplementary Fig. 22B–C). These findings indicate that trainedimmunityenhancestheresponsivenessofthemyeloidcompartmenttoadenoviralstimulation,potentiallyamplifyingdownstreamvaccineeffects.

We then evaluated whether trained PBMCs could enhanceantigen-specific T cell responses to a clinically tested HLA-A2–restrictedCEApeptidevaccine.PBMCsfromHLA-A2⁺donorsweretreatedwithPBSorWPG,stimulatedwithpeptide,andrestimulatedonday 6 (Fig. 8C). Across three independent donors, trained PBMCs-exhibited enhanced IFN-γ responses compared to PBS controls(Fig. 8D–E), confirming that trained immunity augments vaccine-inducedTcellactivationinahumancontext.

Toassessfunctionalrelevanceagainsttumors,wetestedtrainedPBMCs in co-culture with CRC patient-derived organoids (PDOs).PBMCsfromHLA-A2⁺donorsweretrainedwithWPGandexpandedwithCEApeptide,followedbyCD8⁺Tcellenrichmentandco-culturewith PDOs (Fig. 8F). After 24h, microscopy and Z-stack imagingrevealed increased PI staining of tumor cells in PDO#2 and PDO#3whenculturedwithtrainedPBMCscomparedtonon-trainedcontrols(Fig. 8G, H, and Supplementary Fig. 23A). LDH cytotoxicity assaysconfirmedenhancedtumorcellkillingacrossmultiplePDOs(Fig.8I).Importantly,macrophagestrainedwithWPGdidnotexhibitanydirecttumoricidal activity when co-cultured with PDOs (SupplementaryFig. 23B), suggesting that their role is indirect, mediated throughorchestrationofNKandTcellresponses.

To directly test whether macrophages are essential for trainedimmunityinhuman settings,wefirstexamined theirmetabolic andepigeneticchanges.ComparedtoPBScontrols,WPG-trainedmacrophages showed higher glucose uptake (Supplementary Fig. 23C),indicatingmetabolicactivation.ChIP-qPCRfurtherrevealedincreasedH3K4me3 enrichment at the Cxcl9 and Cxcl10 loci (SupplementaryFig. 23D), demonstrating that trained macrophages undergo epigenetic remodeling to boost chemokine expression. Functionally,trainedmacrophagespromotedNKcellmigrationinatranswellassaydependentontheCXCR3axis,consistentwiththeirroleininitiatingtheNK–cDC1axis(SupplementaryFig.23E).

Wenexttestedwhethermacrophagesarerequiredforboostingvaccine-inducedresponses.PBMCsfromHLA-A2⁺donorswereeitherleftintactordepletedofmonocytes/macrophagesbeforeWPGtraining. Both groups were then stimulated with a clinically tested CEApeptidevaccineandtestedinakillingassayagainstHCT116CRCcells.Intact PBMCs trained with WPG exhibited significantly enhancedtumorkilling compared to non-trained controls (SupplementaryFig.23F).Incontrast,PBMCslackingmonocytes/macrophagesfailedtoshowanyincreaseintumorkillingafterWPGtraining,evenwhenthevaccwhenthevaccinepeptidewasprovided(SupplementaryFig.23F).Thisdemonstrates that macrophages areindispensable fortrained immunityinhumans:theyprovidetheinitialreprogrammingsignalthatenablesNKcell recruitment, DC activation, and ultimately stronger CD8⁺ Tcell–mediatedtumorkilling.

Methods
Ethicalpermits
TheiCANFlagshipProjectbiobankstudyisbasedonabiobankconsentandwasreviewedbytheHUSEthicalCommitteeandisexecutedbased on a HUS research permit (4.5.2023 §38 (HUS/223/2023), a Findata data permit (THL/1338/14.02.00/2022), and MTAs with Helsinki Biobank (HBP20210170) and Finnish Hematology and Registry BiobankFHRB(12.5.2022).

AllanimalstudieswereevaluatedandauthorizedbytheExperimentalAnimalCommitteeoftheUniversityofHelsinkiandtheProvincial Government of Southern Finland (permits ESAVI/11895/2019andESAVI/12722/2022).Ourethicalauthorizationlimitstumorsizetoa maximumdiameterof18mm,andthislimitwasnotexceededinanyexperimentconductedforthisstudy.

Murinecelllines
MurinecolonadenocarcinomaCT26(ATCCCat#CRL-2638)waspurchasedfromtheAmericanTypeCultureCollection(ATCC)after2013.Murine colon adenocarcinoma MC38 (Kindly provided by CristianSmerdou, Cima Universidad de Navarra). Murine colon adenocarcinomaColon-2waspurchasedfromCytion(Productnumber:400156).Celllineswerethawedatpassage5andkeptincultureuntilreachingpassage 15. All cell lines were authenticated by the ATCC, culturedunderappropriateconditionsandregularlycheckedformycoplasmacontamination.

Preparationofconditionallyreplicatingadenovirus
All adenoviruses were generated as conditionallyreplicating adenoviruses using standard protocols previously described40,63. In short,adenovirus viruses are of the chimeric 5/3 serotype with a 24-nucleotide deletion and deleteion of the gp19k region in the E1AregionresultinginselectivereplicationinRb-deficientpathwaycells.

PCcomplexformation
The PeptiCab complex was generated by combining the adenoviralvectorwithapeptidethatincludesapolyKtail.TwentymicrogramsofPolyK-extendedepitopeswereincubatedwiththe1×109viralparticlesfor 15minutes at room temperature prior to treatment with theresultingcomplexes.Detailedinformationonthestabilityandformation of the complex has been reported previously41,42. The specificquantitiesofpeptideandvirususedineachexperimentareprovidedinthecorrespondingfigurelegends.

PBMCs,PMNsandmonocytecollection
Polymorphonuclearcells(PMNs)andperipheralbloodmononuclearcells(PBMCs)wereisolatedfrombuffycoatsaspreviouslydescribed65.The PBMC and PMN layers were subsequently removed between serumandFicoll(Cytivia,Cat#1714400)orintheHistopaque(Sigma-Aldrich,Cat#11191)layer,respectively,andculturedin1xRPMI(RoswellParkMemorialInstitute,Gibco,Cat#21875034),

PBMC were processed for monocyte enrichment using densitygradientcentrifugationwithPercoll(Sigma-Aldrich,Cat#P1644)solutions. PBMC suspension (containing approximately 150–200×106 cells)wascarefullylayeredontoahyper-osmoticPercoll.Tubeswerecentrifugedat580gfor15minandcellsattheinterfacewerecollectedand then centrifuged at 350g for 7min. Cell pellet resuspended inRPMImediumtoproduceamonocyte-enrichedsuspension.Tofurtherpurifythemonocytepopulationbyremovingplateletsanddeadcells,10mLof iso-osmoticPercoll solution wasoverlaid with 3mL of themonocyte-enriched suspension (up to 200×106 cells) followed bycentrifugationat350gfor15min.Thesupernatantwasremoved,andcellpelletswereresuspendedinRPMImedium.Samplesweretakenfrom buffy coats from the Finnish Red Cross blood service (ethicalpermit26/2025).

Tumorimplantationandinnateimmunetrainingforanimalexperiments
All animalexperiments were reviewed and approved by the ExperimentalAnimalCommitteeoftheUniversityofHelsinkiandtheProvincialGovernmentofSouthernFinland(licensenumberESAVI/11895/2019).Around4–6-week-oldimmunocompetentfemaleBALB/cmice,purchased from Envigo (Venray, Netherlands), injected in the rightflankwith500,000CT26cells.AsforMC38tumors,C57BL/6micepurchased from Envigo (Venray, Netherlands) were injected in the rightflankwith500,000cells.Allmiceweremaintainedonastandardrodentchowdietprovidedadlibitum.Animalsreceiveddrypelletfeed(Envigo; Catalog No. 2018S) unless otherwise specified. Food andwater were always available throughout the experiments. Tumorvolumewascalculatedusingthefollowingformula:

TumorVolume=longmeasure×ðshortmeasureÞ2 / 2

A complete randomization was performed on the day of thetreatment.Detailsonthescheduleofthetreatmentcanbefoundinthefigurelegends.Theviraldoseusedwas1×109vp/tumorcomplexedwith 20µg of a single peptide. The tumors were measured with acaliperandthemiceweresacrificedwhenthehumaneethicalendpointwasmet.

Forprophylacticsettings,miceweretrainedbyinjectingtwoI.Padministratorsof1mgofWPG(Invivogen,Cat#tlrl-wgp)7and4daysbeforetumorimplantation.Fortherapeuticsettings,micewerefirstimplantedwithtumorsandthenatday4and7post-tumorimplantationwereinjectedwithtwoadministeringof1mgofWPG(Invivogen,Cat#tlrl-wgp)I.P.

For adoptive transfer experiments, macrophages and NK cellswereisolatedfromsplenocytesofdonormice.CD11b⁺cellswerefirstenriched using CD11b magnetic beads (BioLegend, 480109, 5μl/1millioncellsofbiotin,10μl/1millioncellsofnanobeads)andfurtherpurifiedintoF4/80⁺macrophagesusingF4/80magneticbeads(BioLegend, 480169, 5μl/1 million cells ofbiotin, 10μl/1 million cells ofnanobeads).NKcellswereenrichedusingaNKisolationbeads(BioLegend,480049,5μl/1millioncellsofbiotin,10μl/1millioncellsofnanobeads).PurifiedmacrophagesorNKcellswerestimulatedwith1μg/mL WPG for 24h, washed extensively, and subsequently transferredintorecipientmice(1×10⁶cellspermouse,I.P.).

Invivodepletionofspecificimmunecelltypes
Formacrophagedepletion,micewereadministeredi.p.with200µlofchlodronate liposomes (Liposoma, Cat# C-005) 1day after tumorimplantationsand theneveryfour days until theend oftheexperiment.As for NK depletion, miceweretreatedwith50μlofanti-Asialo-GM1antibodies(Purifiedanti-Asialo-GM1Antibody,BioLegend,Cat#146002)dayaftertumorimplantationsandgiveneveryfourdaysuntiltheendoftheexperiment.Fordepletionofbothimmunepopulation,miceweretreatedwithbothtreatments1dayaftertumorimplanationsandgiveneveryfourdaysuntiltheendoftheexperiment.ForCD8+Tcelldepletion,miceweregivenabolustreatmentof500µgofanti-CD8antibody (BioXcell,Cat##BE0061)given I.P 1day prior the firstPCtreatmentandwasfurthersustainedbyinjecting100µgofanti-CD8antibodyI.Pevery3days.

Seahorsemetabolicassay
ASeahorseXFe96Analyzer(Agilent)wasusedtodetermineoxygenconsumptionrate(OCR)andextracellularacidificationrate(ECAR)inmacrophages.MacrophageswereenrichedusingF4/80microbeads(BioLegend,Cat#480169).PurifiedmacrophagesweretransferredtoSeahorse XF Base medium (Agilent) supplemented with glucose(10mM),sodiumpyruvate(5mM),andL-glutamine(5mM)(Gibco),pH7.4,at37°C.Cellswereplatedateither0.5×105or0.8×105cellsper well onto Seahorse XF96 cell culture plates and allowed toadhere.Macrophageswerethenstimulatedwitheither1µg/mlwholeβ-glucanparticles(WPG;InvivoGen,Cat#tlrl-bgp)orPBScontrolfor either 2h (short-term stimulation) or overnight (16h; long-termstimulation).

OCRandECARweremeasuredusingastandardMitoStressTest.Thefollowinginhibitorsweresequentiallyinjectedduringtheassay:oligomycin (1µM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone(FCCP;1µM),andamixofantimycinAandrotenone(1µM each).Assayparameterswere3minmixing,nowait,and3minmeasurement,repeated3timesatbaselineandaftereachinjection.Following measurements protein determination was performed, andresultswerenormalizedfortotalproteincontent.

Creatineinhibition
Mice received I.P injection of β-GPA (500mg/kg in 100μlf PBS)(Sigma-Adlrich,Cat#G6878)1dayaftertumorimplantationsandgiveneverydayuntiltheendoftheexperiment.

Intracellularstimulationandstaining
Intracellularstimulationandstainingwereperformedon2×106murinesplenocytesincubatedina96-wellU-bottomplatewithbrefeldinA(Biolegend,Cat#420601)(1:1,000)andmonensinA(Biolegend,Cat#420701) (1:1,000) at 37°C for 5h. Stimulants were added at concentrationsof2µgforpeptidesand40MOIforadenovirus.Followingstimulation,sampleswerestoredovernightat4°C.Thecellswerethencentrifuged and the pellets were resuspended in murine Fc-block(BioLegend,Cat#156613)at4°Cfor15minutes.Extracellularproteinswere stained using the following antibodies: BV711 CD3 (BD Biosciences, #563123,0.5μl/1 million cells), BV510 CD8 (BD Biosciences,#563068,1μl/1millioncells),PE-CF594CD4(BDBiosciences,#562285 0.5μl/1 million cells), APC CD62L (eBioscience, #17-0621-81 0.5μl/1millioncells),AF700CD44(BioLegend,#103026,1μl/1millioncells),andv450CCR7(BDBiosciences,#560805,1μl/1millioncells).Afterextracellularstaining,thecellswerefixedandpermeabilizedusingaFixation/Permeabilizationkit(BDBiosciences)accordingtothemanufacturer’sinstructions.IntracellularproteinswerethenstainedwithBV650 TNF-α (BioLegend, #506333, 1μl/1 million cells), PE-Cy7 IL-2(BioLegend, #503832, 1μl/1 million cells), PE IFN-γ (BioLegend,#505808,1μl/1millioncells),andFITCIL-6(BioLegend,#503806,1μl/1millioncells).Finally,thesampleswereacquiredonaFortessaflowcytometer(BDBiosciences)andanalyzedwithFlowJosoftware(version10.8.2).

Flowcytometryoftumorsamples
Tumorsfrommicewerepassedthrougha70µmcellstrainer(Falcon)toobtainacellsuspension.Onemillioncellswerethenplatedina96Vbottomwellplate(Sarstedt)andstainedwiththefollowingantibodies:BV711CD3(clone:145-2C11,cat:563123,BDBiosciences,0.5μl/1millioncells), BV510 CD8 (clone: 53-6.7, cat: 563068, BD Biosciences, 1μl/1million cells), PE-CF594 CD4 (clone: RM4-5, cat: 562285, BD Biosciences,0.5μl/1millioncells),APCCD62L(clone:MEL-14,cat:17-0621-81,eBioscience,0.5μl/1million cells),AF700CD44(clone:IM7,cat:103026,BioLegend,1μl/1millioncells),v450CCR7(clone:4B12,cat:560805,BDBiosciences,1μl/1millioncells),BV650TNF-α(clone:MP6-XT22,cat:506333,BioLegend,1μl/1millioncells),PE-Cy7IL-2(clone:JES6-5H4,cat:503832,BioLegend,1μl/1millioncells),PEIFN-γ(clone:XMG1.2, cat: 505808, BioLegend, 1μl/1 million cells), BV711 CD11b(clone: M1/70, cat: 101241, BioLegend, 0.5μl/1 million cells), FITCCD11c(clone:N418,cat:25-0114-82,eBioscience,0.5μl/1millioncells),PerCP/cy5.5H2-Kd(clone:AF6-88S,cat:116516,BioLegend,1μl/1millioncells),PECD103(clone:2E7,cat:121406,BioLegend,1μl/1millioncells), APC PDCA (clone: 927, cat: 127015, BioLegend, 1μl/1 millioncells), APC anti-mouse CD3 (clone: 17A2, cat: 100236, BioLegend,0.5μg/1 million cells), FITC anti-mouse CD8a (clone: 53-6.7, cat:100706,BioLegend,1μg/1millioncells)andPerCP/cy5.5anti-mouseCD107a(LAMP-1)(clone:1D4B,cat:121625,BioLegend,5μl/1millioncells),Allantibodymixeswereincubatedinafinalvolumeof100μlandgenerally used according to their respective manufacturerinstructions.

In-vitrohumanimmunecelltrainingandstimulation
100,000purifiedmonocyteswereresuspendedinavolumeof100μLwasdispensedintoflat-bottom96-wellplates(Corning,NY,USA)andallowedtoadheretothepolystyrenesurfacefor1hat37°C.Afterward,cellswerewashedwithpre-warmed.MonocyteswerethenincubatedwitheitherRPMIculturemediumasanegativecontrolorwith1μg/mLofβ-glucanat37°Cfor24h.After,cellsweretreatedwithbrefeldinA(1:1,000)andmonensinA(1:1,000) at37°Cand stimulated with 40MOIofadenovirusesorLPS(Invivogen,Cat#,tlrl-3pelps)(10ng/ml)for5h. As for macrophages, monocytes were differentiated into M0macrophages by supplementing RPMI mediawith 10% humanheat-inactivatedserumforfivedays.ForDCs,monocyteswerestimulatedwith GM-CSF (1000U/ml) and IL-4 (800U/ml) for 4 days. DCs andmacrophageswerethenincubatedwitheitherRPMIculturemediumasanegativecontrolorwith1μg/mLofβ-glucanat37°Cfor24h.After,cellsweretreatedwithbrefeldinA(1:1,000)andmonensinA(1:1,000) at37°Candstimulatedwith40MOIofadenovirusesorLPS(Invivogen,Cat#,tlrl-3pelps)(10ng/ml)for5h.

Ex-vivometabolomicinhibiton
Balb/cmicewerefirsttrainedwitheitherPBSorWPGandthensacrificed.Spleenswereisolatedandwerethenpassedthrougha70µmcellstrainer(Falcon)toobtainacellsuspension.CD11b+cellswereisolatedusingCD11bmagneticbeads(BioLegend,480109,5μl/1millioncellsofbiotin,10μl/1millioncellsofnanobeads)andplatedindispensedinto flat-bottom 96-wellplates and allowed to adhere. CD11b+ cells werethen treated with 10nM rapamycin (Sigma), 500nM of ascorbic(Merck,Cat#APO456787373)and10mMofβ-GPAfor5daysandthenwashedwithPBS.CD11b+cellswerethenstimulatedwithPBSor40MOI of adenoviruses for 5h. For TNF-α (BioLegend, 502909, 1μl/1millioncells)andIL-6measurement-α(BioLegend,501112,1μl/1millioncells),CD11b+ cellsweretreatedwithbrefeldinA(1:1000)andmonensinA(1:1000)tomeasurelevelswithintracellularflowcytometry.Asfor CXCL9 and CXCL10, the concentrations of each cytokine wasdeterminedinthecellculturesupernatantbyELISAusingMIG/CXCL9(ThermoFisher,Cat#250-18-20UG)andmurineCXCL10/IP-10ELISAkit(R&D,Cat#DY466-05).

ImmunofluorescenceonF4/80macrophages
Tumorsfrommicewerepassedthrougha70µmcellstrainer(Falcon)toobtainacellsuspension.CD11b+cellswereisolatedusingCD11bmagneticbeads(BioLegend,480109,5μl/1 million cells of biotin,10μl/1millioncellsofnanobeads)andthenfurtherenrichedforF4/80macrophagesusingF4/80beads(Biologend,480169,5μl/1million cells of biotin, 10μl/1 million cells of nanobeads). F4/80macrophagecellswereplatedon8wellNunc;Lab-Tek;IIChamberSlidesandculturedfor4days.Cellswerefixedin4%coldparaformaldehydeandstainedwithH3K4me3antibody(1:200,Diagnode,C15410003)and DAPI (1μg/ml, Invitrogen™, P36941) Microscopy pictures weretaken using an EVOS FL cell imaging system (Thermo FisherScientific).

Transwellassay
Tumorsfrommicewerepassedthrougha70µmcellstrainer(Falcon)to obtain a single-cell suspension. CD11b⁺ cells were isolated usingCD11bmagneticbeads(BioLegend,480109)andfurtherenrichedforF4/80⁺macrophagesusingF4/80beads(BioLegend,480169),NKcellusingthemouseNKcellisolationkit(BioLegend,480049,5μl/1million cells of biotin, 10μl/1million cellsof nanobeads) orDCs usingMouse Pan Dendritic Cell Isolation Kit (BioLegend, 480097, 10μl/1millioncellsofbiotin,10μl/1millioncellsofnanobeads).

For murine macrophage–NK migration assays, F4/80⁺ macrophageswereplatedinthebottomchamberandstimulatedwith40MOI of adenovirus. NK cells were fluorescently labeled with 5μmCellTracker™ Green CMFDA Dye (Thermo Fisher Scientific,Cat#C2925)andseededintheupperchamberof6.5mmTranswell®polycarbonate membrane inserts with 5.0µm pore size (Corning).Whereindicated,NKcellswerepre-incubatedwith10µg/mlofanti-mouse CXCR3 antibody (BioXcell, Cat#BE0249) prior to migration.After24h,migratedNKcellswerequantifiedusingaBDLSRFortessa(BD Biosciences) with Precision Count Beads™ (BioLegend,Cat#424902,5μlpersample).

FormurineNK–DCmigrationassays,NKcellswereisolatedfromthe TME using the NK cell isolation kit (BioLegend, 480049, 5μl/1millioncellsofbiotin,10μl/1millioncellsofnanobeads),whileDCswereisolatedusingtheMojoSort™MousePanDendriticCellIsolationKit(BioLegend,480097,10μl/1millioncellsofbiotin,10μl/1millioncellsofnanobeads).Whereindicated,NKcellswerestimulatedwith1µg/mlofWPGovernightandthenwashedwithPBS.DCswerelabeledwith5μmCellTracker™GreenCMFDADyeandplacedintheupperchamber of Transwell® inserts (5.0µm). NK cells were plated in thelowerchamberandstimulatedwith40MOIofadenovirus.Inselectedconditions,NKcellsweretreatedwith10µg/mlofanti-CCL5antibody(BioXcell,Cat#BE0449)priortomigration.After24hours,migratedDCs were quantified by flow cytometry on a BD LSRFortessa usingPrecisionCountBeads™.

ChIP-qPCRonmurinesamples
Chromatin immunoprecipitation (ChIP) was performed to assessH3K4me3 enrichment at target loci. Briefly, 1×107 cells were crosslinked inculture with 0.75%formaldehyde for 10minatroom temperature,followedbyquenchingwith125mMglycinefor5min.Cells werewashedtwiceincoldPBS,collectedbyscraping,pelleted,andresuspended inFAlysisbuffer (750µl per 1×10⁷ cells).Nuclei weredisrupted by sonication for 15 cycles (1min ON and 1min OFF) formurinesampleswhileforhumansamples25cycles(1min ON and 1min OFF) to generate chromatin fragments of 500–1,000bp, confirmedon a 1.5% agarose gel. Input samples were saved prior toimmunoprecipitation.

ForChIP,25µgproteinperreactionwasdiluted1:10inRIPAbufferandincubatedovernightat4°Cwithanti-H3K4me3antibodyorwithcontrolIgGantibody(1µgantibodyper25µgprotein)andProteinA/Gbeads (pre-blocked with herring sperm DNA and BSA). Bead-onlysamplesservedasnegativecontrols.Beadsweresequentiallywashedthreetimeswithwashbufferandoncewithfinalwashbufferbeforeelutionin120µlelutionbuffer.Cross-linkswerereversedat65°Cfor4–5h with RNase A and proteinase K treatment. DNA was purifiedusingaPCRpurificationkit(Qiagen)accordingtothemanufacturer’sinstructions.

Purified DNA was analyzed by quantitative PCR using primersspecificfortargetpromoters(e.g.,CXCL9,CXCL10,Slc6a8).CtvaluesfromimmunoprecipitatedsampleswerefirstcorrectedbysubtractingIgGbackground.DatawerethennormalizedtoinputDNAtoaccountfor differences in chromatinquantity. Enrichment was expressed asfoldenrichmentrelativetocontrolsamples(non-trained).PrimercanbefoundinSupplementaryTable1.

RT-qPCR
NKcellswereisolatedfrommousetumorsusingtheMojoSort™MouseNK Cell Isolation Kit (BioLegend, Cat#480049) according to themanufacturer’s instructions. Total RNA was extracted using theRNeasy Mini Kit (Qiagen, Cat#74104), and RNA concentration andpurity were determined using a NanoDrop spectrophotometer(Thermo Fisher Scientific). For cDNA synthesis, RNA sample wasreversetranscribedusingMaximaReverseTranscriptase(200U/μL;Thermo Fisher Scientific, Cat#EP0742) following the manufacturer’sprotocol.

Quantitative PCR (qPCR) was performed using GoTaq® qPCRMasterMix(Promega,Cat#A6002)onaQuantStudio5Real-TimePCRSystem(AppliedBiosystems).Eachreactionwasrunintechnicaltriplicateswiththefollowingcyclingconditions:95°Cfor2min,followedby40cyclesof95°Cfor15sand60°Cfor1min.ExpressionlevelswerenormalizedtoGapdh,andfoldchangewascalculatedusingtheΔΔCtmethodrelativetoPBS-treatedNKcellcontrols.PrimercanbefoundinSupplementaryTable1.

Long-termgenerationofpeptide-specificCD8+Tcells
IsolatedPBMCsweretrainedusingPBSor1μg/mLofWPGfor24h.Trained PBMCs were cultured with GM-CSF (1000U/mL) and IL-4(800U/mL)for4daystodifferentiateintomonocyte-deriveddendriticcells (moDCs). To stimulate peptide-specific T cells, PBMCs werepulsedwithPC(10μMofpeptidescomplexedwith109viralparticles).Subsequently,TNFα(10ng/mL,Peprotech)andLPS(10ng/mL)weredirectlyappliedwhichwereincubatedfor4htogeneratesemi-maturedendritic cells. After, IL-21 (60ng/mL, Peprotech) was added tofurther T-cell expansion. After 10–12 days, the T cells were restimulated with PC for an additional 10 days. The cultures weremaintainedbyfeedingevery2–3dayswithIL-2(50U/mL,STEMCELLTechnologies).

Short-termgenerationofpeptide-specificCD8+Tcells
106PBMCswereplatedina24-wellplateandtreatedwithPBSor1μg/mLofWPGfor24h.CellswerethenwashedandstimulatedwithPC(10μM of peptides complexed with 109 viral particles). After threedays,IL-2 (50U/mL, STEMCELL Technologies) wasadded to furtherexpand T cells.PBMCs werethen restimulated with PC and treatedwithbrefeldinA(1:1,000)andmonensinA(1:1,000)at37°Cfor5h.

Microscopyandquantification
Microscopy visualization of PDO killing was conducted with EVOS conducted with EVOSM7000 Imaging System (Thermo Fisher Scientific)whereimages ofgreenlabeledCD8+T-cellsandcelldeath(usingPI)wereobtainedinthetransmitted,GreenFluorescentProtein(GFP)andRedFluorescentProtein(RFP)channels respectively. Settings including brightness,focus and counts per image were established manually, with thedesiredtargetsperimage.Aftermanualcalibrationautomatedimageacquisitionwasperformedtoobtain50imagesperwellacrossmultiplechannelsandbuildaZ-stack.Automatedimageacquisitionforthisstudy included transmitted (for conventional colored precipitateproduct),RFPandGFP.

Metabolomicprofiling
SortedCD11b+cellswereaddto400μlofcoldextraction solvent, followed immediately by centrifugation at14,000rpmfor5minutesat4°C.Thesupernatantswerethenloadedonto an Ostro 96-well plate (25mg; Waters) and processed using arobotic vacuum. The resulting filtrates were transferred into polypropylenetubesanddriedcompletelyinanitrogengasevaporator.Thedriedresidueswerereconstitutedin40μlofextractionsolvent,vortexed for 2minutes, and then transferred into HPLC glass autosampler vials. Two microliters of each sample were injected into aThermoVanquishUHPLCsystemcoupledwithaQ-ExactiveOrbitrapquadrupole mass spectrometer fitted with a heated electrosprayionization(H-ESI)source(ThermoFisherScientific).Chromatographicseparation was performed using a SeQuant ZIC-pHILIC column(2.1mm×100mm,5μmparticlesize;Merck)undergradientelutionata flow rate of 0.100ml/min. Mobile phase A, composed of 20mMammonium hydrogen carbonate adjusted to pH 9.4 with a 25%ammoniumsolution,andacetonitrileasmobilephaseBwereusedinthefollowinggradient:startingat20%Aand80%B,maintaineduntil2minutes, then gradually increasing mobile phase A to 80% by17minutes,andfinallyreducingbackto20%Aat17.1minutes,whichwas held until 24minutes. The column oven and autosampler weremaintainedat40°±3°Cand5°±3°C,respectively.Massspectrometrywasperformedwithpolarityswitchingunderthefollowingconditions:a resolution of 35,000; spray voltages of 4250V (positive mode) and3250V(negativemode);sheathgasat25arbitraryunits;auxiliarygasat 15 arbitrary units; sweep gas flow of 0; capillary temperature at275°C;andanS-lensRFlevelof50.0.TheinstrumentwasoperatedusingXcalibur4.1.31.9software(ThermoFisherScientific),andmetaboliteswereannotatedfor462metaboliteswithTraceFinder4.1software (Thermo Fisher Scientific) using mass to charge ratios andretentiontimeofstandards(MSMLS-1EA,Merck).Throughouttherun,thedataqualitywasmonitoredbyincludingapooledserumqualitycontrol (QC) sample every 10th injection. The final data was pre-filteredformetaboliteshavingpoorchromatograph,highvariationinpooled QC samples >20% RSD or background noise >20% whencomparedtoblanksamples.DatawasanalysedwithMetaboAnalyst5.0(https://www.metaboanalyst.ca/).Missingvariableswereimputedbyreplacingwith1/5oftheminimumpositivevaluesofindividualfeatures.Thedatawasfurthernormalizedwithlog10-transformationandauto-scaling. In multivariate clustering analysis the heatmap wascomputed using distance measureeuclideanforup to 25 mostsignificantfeatures(t-test).Principalcomponentanalysis(PCA)wasusedto visualize the data.For Univariate analysis t-test and volcanoplotanalysiswereperformedwithfoldchange1.5andrawp-value<0.05.Quantitative metabolite set enrichment analysis (MSEA) was performedintheMetaboAnalyst5.0usingthemouseKEGGdatabase.

HashtingsamplesforSingle-cellRNAsequencing
SortedCD11b+cellswerewashed2–3timeswith10mLofPBSandthenresuspended in 100μL of Cell Staining Buffer (BioLegend, Cat#420201). Next, 10μL of TruStain FcX blocking reagent (BioLegend,Cat#420510)wasadded,andthecellswereincubatedfor10minutestoblocknon-specificbinding.AuniqueTotalSeq-Ahashingantibody(BioLegend, Cat#1603-1608, 1μl/1 million cells) was then added toeachsampleataconcentrationof1–2μL(or1–2μgpersample)andincubatedfor30minat4°Cinthedark.Followingthis,thecellswerewashed3–5timeswith3mLofstainingbuffer,combinedintoasingle1mLvolumeofstainingbuffer,centrifuged,andfinallyresuspendedinPBScontaining0.04%bovineserumalbumin(BSA)beforeproceeding toscRNA-seq.

Single-cellRNAsequencing
TheChromiumSingleCell3’RNAseqrunandlibrarypreparationswereperformedusingthe10xGenomicsChromiumNextGEMSingleCell3’Gene Expression v3.1 DualIndex chemistry, with modifications outlined by Stoeckius et al.22 (https://cite-seq.com/) and based on anupdated protocol available at www.biolegend.com/en-us/protocols/totalseq-a-antibodies-and-cell-hashing-with-10x-single-cell-3-reagent-kit-v3-3-1-protocol. Approximately 15,000 cells were aimed perlane (GEM well) that contained hashed samples from 13 cell lines,whichwereculturedeitheraloneorincombinationwithNKcells,withseparate GEM wells allocated for each treatment group. This setupresulted in targeting roughly 1000–1200 cells for sequencing fromeach cell line in mono- or co-culture. The 3’ GEX and Cell Hashing(multiplexing) libraries were sequenced on an Illumina NovaSeq6000system,withreadlengthssetat28bpforRead1,10bpforthei7Index,10bpforthei5Index,and90bpforRead2.

Single-CellRNAsequencinganalysis
scRNA-seqdatawereprocessedusingSeurat(v6.0)inR(v4.3.2).Forquality control, cells were filtered to exclude those with <200 or>2500detectedgenes,>30%mitochondrialgenecontent,orgenesexpressed in fewer than three cells. Cells with abnormally highribosomal content (>50%) or low UMI counts (<700) were alsoremoved.

GeneexpressionvalueswerenormalizedwiththeNormalizeDatafunction using the LogNormalize method (scaling factor = 10,000).Highlyvariablegenes(HVGs;n=2000)wereidentifiedusingthe“vst”methodinFindVariableFeatures.DatawerescaledandcenteredusingtheScaleData function.To controlfor cell-cycle heterogeneity,cell-cyclescoreswerecomputedwithCellCycleScoring(G2/MandSmarkersprovidedinSeurat)andregressedoutduringscaling.

Dimensionalityreductionwasperformedbyprincipalcomponentanalysis(PCA)onHVGs.Thetop30PCswereselectedbasedonelbowplotinspectionandJackStrawresampling.Tomitigatebatcheffectsacross experiments, we applied Harmony (v1.0) with default parameters,usingsampleidentityasthecovariate.

Clustering was performed using the FindNeighbors andFindClustersfunctions.Forglobalclusteringacrossallimmunecells,weusedaresolutionof0.5;forlymphoidcell-focusedsubclustering,aresolution of 0.5 was applied; and for TAM/monocyte analyses, weused 0.4. Two-dimensional visualization was performed with UMAP(RunUMAP,30PCs,defaultsettings).

Cluster annotation was guided by differentially expressed (DE)markers,identifiedwiththeFindAllMarkersfunction(Wilcoxonrank-sum test, log2 fold-change >0.8 or <−0.8, adjusted pvalue<0.05,Bonferronicorrection).DifferentialexpressionbetweenexperimentalgroupswasperformedwiththeFindMarkersfunctionusingthesamethresholds.Celltypeidentitieswerethenassignedbymanualreviewofcluster-specific DEGs and confirmed using the ImmGen MyGeneSettool,ratherthanautomatedclassificationtools,toensurebiologicallygroundedannotations.

TissuecollectionforPDOcultureandethicalperimits
Tissuesampleswereobtainedbysurgeoneitherviaendoscopicbiopsyforceps or via biopsy from surgical specimen. The samples weretransported to the laboratoryin a transfer medium (DMEM supplemented with 1% Penicillin-Streptomycin solution) and processedwithin24haftercollection.TheiCANFlagshipProjectbiobankstudyisbased on a biobank consent and was reviewed by the HUS EthicalCommitteeandisexecutedbasedonaHUSresearchpermit(4.5.2023§38(HUS/223/2023),aFindatadatapermit(THL/1338/14.02.00/2022),andMTAswith HelsinkiBiobank(HBP20210170)andFinnish HematologyandRegistryBiobankFHRB(12.5.2022).Allsamplesusedinthisstudyweredestroyedaftertheanalysis.

PDOculture
TissuesamplesweredissociatedmechanicallybyP1000pipette,followed,ifnecessary,byaten-minuteenzymaticdigestionwithOrganoidDigestionMediumtoincreasebiomassoutput.Dissociatedcellswere washed 1 or 3 (in case of enzymatic digestion) times by centrifugation (5min at 4°C, 200g) in Wash medium, resuspended inMatrigel(Corning,Cat#354230)andplatedasasingledomes(50µLeach)ona24-wellplate(1dome/well).AfterMatrigelsolidifying(15-30minin37°C),500µLofOrganoidCultureMediumsupplementedwith1µL/mLRho-associatedcoiled-coilkinaseinhibitorY-27632wasaddedtocoverthedomes,andplateswerereturnedto37°C(with21%O2and5%CO2)andculturedforexpansion.OrganoidCultureMediumwaschangedevery7days.Organoidswerepassagedevery14days(or7daysifnecessary).

T-cellkillingassayswithPDOs
CRC PDOs were co-cultured with 100,000 expanded CD8+T-cells.CD8+T cells were injected using a 27G needle straight into thematrigel. Propidium iodide was added (1:1000) to the culture tovisualizedeath.After24hofincubationat37°C,cellkillingwasmeasuredbydeterminingtheamountofLDHreleasedusingacolometricassay(CyQUANTLDHCytotoxicityAssay,Cat#C20303).Specificlysiswasthencalculatedusingthefollowingformula:Percentcytotoxicity=(“experimental”–“target spontaneous”)/(“target maximum”–“targetspontaneous”)×100%,where“experimental”correspondstothesignalmeasuredinatreatedsample,“targetspontaneous”correspondsto the signal measured in PDOsaloneand“targetmaximum”correspondsto the signal measured in the presence of detergent lysedtumorcells.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript entitled, \"Leveraging trained immunity for the epigenetic and metabolic rewiring of macrophages to enhance colorectal cancer vaccination by inducing an NK-cDC1 axis\" reported that combined trained immunity and tumor vaccine led to significant tumor killing using both mouse colon cancer models and human colon cancer organoids. The authors showed that the combined trained immunity with vaccine therapeutic efficacy appears to be dependent on a robust macrophage-NK cell-cDC axis. While these results have clear clinical translational potential, there are some concerns in this study.\n1. The role of cDC1 is unclear in this study. While the authors showed that cDC1 were accumulated in the tumor-bearing mice treated with trained immunity plus PC vaccine and its correlation with enhanced T cell activity (Figure 2), the direct evidence to show that cDC1 accumulation is due to trained macrophages is lacking. In addition, cDC1 in mice normally do not express CD11b yet Figure 4 scRNAseq data showed cDC1 cluster using sorted CD11b+ cells from tumors.\n2. The role of T cells. The authors depleted macrophages and NK cells to demonstrate that trained immunity+PC therapeutic efficacy was dependent on macrophages and NK cells (Figure 6). The authors should also deplete CD4/CD8 T cells to determine the relative contribution of innate antitumor immunity and T cell antitumor immunity in animal tumor models.\n3. Figure 1A-E. These publicly available datasets are from mouse pancreas but not from spleen. The author should use these data in caution as some of myeloid cell subsets may be tissue specific.\n4. Figure 2. The authors analyzed DCs and T cells from draining lymph nodes. The authors should also provide these data from tumors. In addition, it is unclear whether these myeloid cells have \"trained immunity\" phenotype.\n5. Figure 3. It is nice to show metabolomics data. The authors may want to validate some of these pathways. For example, performing Seahorse assay to see whether indeed glycolysis pathway is enhanced in trained myeloid cells.\n6. NK cells. The authors need to examine the functionality of NK cells, such as granzyme and perforin secretion.\n7. Figure 8. The authors may use trained monocytes cultured with PDOs to see whether trained myeloid cells also have direct cancer killing activity.", "reviewer_2": "This study found that β-glucan-induced \"trained immunity\" enhances CRC vaccine efficacy by epigenetically and metabolically reprogramming macrophages. Mechanistically, trained immunity shifts CD11b+ myeloid cells toward aerobic glycolysis, while creatine metabolism provides the additional ATP required for sustained cytokine and chemokine production. This reprogramming converts immunoregulatory macrophages into anti-tumor effectors that recruit NK cells via CXCL9 and CXCL10, thereby enhancing cDC1 function. Finally, using human PBMC and patient-derived organoid co-culture models, they found that trained immunity enhance antigen-specific responses and tumor cell killing. This study presents a promising approach to overcoming the limitations of conventional immunotherapies in CRC by leveraging β-glucan-induced trained immunity to enhance vaccine efficacy. The mechanistic explanation is interesting but many statements/hypotheses are short of experimental evidence support. Specifically, several issues remain unresolved:\n1. Figure 2B showed slightly enhanced anti-tumor efficacy of vaccination after β-glucan training, while Figure 2M is the result of a combination therapy approach. But for the same “Mock” group, the tumor growth is faster in therapeutic setting than the prophylactic setting. Did the mice receive the same amount of CT26 cells in both settings?\n2. In Extended Figure 4, the authors showed tumor growth curves for 8 mice, but for the statistical analysis of dendritic cell phenotype in draining lymph nodes, they selected only 3 mice per group (Figure 2C). It's unclear whether these results come from three independent experiments. The same issue appears in Figures 2M-N. The authors need to describe their experimental methods in detail.\n3. Figure 2D, the authors need to provide statistical results. From the presented data, the cell numbers in each group appear inconsistent.\n4. Figure 3, the authors show broad alterations in cellular metabolism caused by different treatments, but these data contain speculative interpretations without solid evidence. For example: Lines 250-252: ECAR and OCR analysis would be desired to support their claims. Lines 277-279: Could the authors use isotope-labeled glutamate to validate the glutamate pathway as a metabolic hub? Although previous studies have shown glutamine is important for trained immunity, glucose metabolism is more widely recognized as the metabolic foundation of trained immunity (PMID: 27866838, PMID: 25258083). The authors need direct evidence to support their hypothesis.\n5. Line 347 appears to describe results from Figure 4H, not 3H.\n6. Similar to Figure 3, Figure 4 seems more descriptive and speculative without substantial experimental evidence. For example, why didn't the authors use CXCL9/10 blocking antibodies to confirm that trained macrophages recruit NK cells via CXCL9/10 (lines 347-349)? Is there direct evidence that NK cells recruit cDC1 cells by secreting CCL5/4 (lines 354-358)? Although Figures 4A-C show increased NK and DC cell numbers in the Trained+PeptiCrad group, these results appear to reflect overall changes in the immune cell landscape. Why did the authors consider NK and DC cells particularly important in this process? Although Figure 6B shows that depletion of macrophages and NK cells significantly weakens the anti-tumor effects of Trained+PC, would depletion of either macrophages or NK cells alone also promote tumor progression?\n7. The authors consider trained macrophages as key immune cells in the NK-DC axis for anti-tumor effects, why does macrophage depletion alone show much weaker anti-tumor effects compared to simultaneous depletion of both macrophages and NK cells (Figure 6B)? Extended Figure 10 shows effective macrophage depletion. The authors need to clarify whether macrophages and NK cells have an upstream-downstream regulatory relationship or a parallel relationship. Trained neutrophils also have anti-tumor functions (PMID: 33125892). Did the authors consider this in their study?\n8. Figure 4C, it's unclear whether this represents quantitative results from Figure 4B, but the proportion of cluster 20 in Figure 4C seems inconsistent with Figure 4B. The color for cluster “0” is almost same as cluster “20”. Please modify the color to better differentiate them.\n9. In Figure 5, the authors propose potential mechanisms regulating trained immunity, but I think the experimental design is confusing. The authors suggest creatine regulates trained immunity, but the author add rapamycin (mTOR/glycolysis inhibitor), ascorbic acid (HIF-1α inhibitor), and 3-guanidinopropionic acid (GPA; creatine uptake inhibitor) during rest and secondary adenovirus stimulation rather than during WPG training (Figure 5A). Classical trained immunity inhibition is typically administered during training (Such as: PMID: 25258083, PMID: 29328908). The authors' experimental design can only show that rapamycin, ascorbic acid and GPA inhibit effector functions during secondary stimulation, not the priming stage when the immune memory undergoes induction during primary stimulation. The results in Figure 5I seem to confirm my suspicion, since 3-GPA didn't inhibit H3K4me3 modification levels, indicating that 3-GPA likely doesn't inhibit trained immunity in myeloid cells but rather inhibits effector immune responses during PC treatment.\n10. If the authors want to firmly prove their conclusion (lines 422-423), I suggest transplanting bone marrow cells or splenic monocytes/macrophages from mice treated with WPG +3-GPA into recipient mice to observe whether they can enhance the anti-tumor effects of PC.\n11. As the authors describe, \"trained immunity is driven by long-lasting epigenetic modifications.\" Previous studies also consider epigenetics as the core mechanism of innate immune memory (PMID: 25258085). In my opinion, the genome-wide H3K4me3 modification analysis lacks precision, and the result complemented with ChIP-seq or ChIP-qPCR validation of relevant gene H3K4me3 modifications would be more accurate.\nMinor issues:\n1. The manuscript contains several errors that need correction: For example: Line 85: \"TAMS\" should be \"TAMs\". Line 132: “SINFEKL” should be “SIINFEKL”; Line 866: \"PBMcs\" should be \"PBMCs\". The authors should carefully proofread the manuscript.\n2. It is suggested that the authors provide some technical background information or brief introduction about the “PeptiCrad (PC)” vaccine platform.\n3.The Figure layout appears unpolished, with inconsistent magnification scales and font sizes among subpanels within the same Figure. The authors should revise these elements to enhance readability.", "reviewer_3": "The article by Hamdan and colleagues reports that β-glucan-induced macrophage trained immunity activates downstream NK cells, DCs and T cells. They conducted studies using the CT26 CRC model and validated their findings in the MC38 CRC model. They have also tried to validate their study in the human CRC organoid system. The study is comprehensive and novel, and most of the experiments are rigorous. Below are some comments that should be addressed:\n1.The authors do not experimentally demonstrate how NK cells act on DCs. FLT3L is mentioned as a factor that can be produced by NK cells to activate DCs in their system. However, NK cells may not be the major source of FLT3L. If the authors believe that FLT3L produced by NK cells plays a role in DC activation, they should compare its expression in NK cells with that in other cell types, such as stromal cells, endothelial cells, and T cells. Then, they should design experiments to prove the role of FLT3 produced by NK cells in activating DCs. If they propose that other molecules such as CCL5 produced by NK cells are involved, the same comments regarding FLT3L also applies.\n2.How long does the trained immunity last?\n3.Does trained immunity induce cell exhaustion of immune cells, such as NK cells?\n4.In the section on human studies, NK cells are not included. This does not recapitulate the mouse studies and also does not align with the title, which refers to “...enhancing colorectal cancer vaccination by inducing an NK-cDC1 axis.”\n5.Figure 6 should include a tumor-only group without any treatment.", "reviewer_4": "The study by Hamdan et al. explores the potential of β-glucan-induced trained immunity to enhance colorectal cancer (CRC) vaccination by modulating macrophage metabolism and promoting an NK-cDC1 axis. While the work addresses an important gap in CRC immunotherapy, its impact is limited by concerns regarding novelty, mechanistic depth, and experimental rigor. Below is a detailed critique organized by section.\nMajor Critiques\n1. Novelty and Overlap with Prior Studies\n- Trained Immunity in Cancer: The use of trained immunity as a cancer vaccine adjuvant has been previously demonstrated, notably by Wattenberg et al. (2023, Sci Immunol), who reported synergistic effects of β-glucan with CD40/Dectin-1 agonists in pancreatic cancer. The current study does not sufficiently differentiate its findings from this prior work.\n- NK-cDC1 Axis: The mechanistic role of NK cells in recruiting cDC1s via CCL5/XCL1 is well-established (Böttcher et al., 2018, Cell). The authors should clarify how their results extend beyond these known mechanisms.\n- Creatine Metabolism: Although the link between creatine metabolism and immune cell function is an emerging area, Peng & Saito (2023, Front Immunol) recently reported similar findings in macrophages, raising questions about the originality of this aspect.\n2. Mechanistic Gaps\n- Epigenetic vs. Metabolic Contributions: The assertion that trained immunity is driven by \"epigenetic and metabolic rewiring\" lacks robust support. While Chil3+ Ly6Chi macrophages are implicated in the NK-cDC1 axis, the specific molecular mechanisms (e.g., how H3K4me3 regulates CXCL9/10 or creatine metabolism) remain unclear. Functional validation (e.g., ChIP-seq, CRISPR-edited models) is needed to strengthen these claims.\n- Causality in the NK-cDC1 Axis: Depletion experiments (Fig. 6) do not conclusively determine whether NK cells or macrophages are the primary drivers of cDC1 recruitment. Transwell assays (Fig. 6I) demonstrate macrophage-NK chemotaxis but fail to directly link this to cDC1 priming.\n- Metabolomic Data Interpretation: The observed upregulation of creatine metabolites (Fig. 3C) is correlative. Isotope tracing or genetic knockout experiments are needed to establish a causal relationship between creatine metabolism and cytokine production. Additionally, does creatine supplementation rescue the effects of β-GPA inhibition in vivo?\n3. Experimental Design and Rigor\n- Mouse Model Limitations: The use of immunogenic models (CT26, MC38) may not fully recapitulate the immunosuppressive tumor microenvironment (TME) of human CRC. Including syngeneic models with low mutational burden (e.g., APC-mutant) would improve translational relevance. Furthermore, subcutaneous tumors do not adequately model metastatic CRC; orthotopic models would be more informative.\n- Inducer Specificity: Relying solely on β-glucan (WPG) as a trained immunity inducer limits generalizability. Comparisons with other inducers (e.g., BCG, LPS) would strengthen the conclusions.\n- Human Data Limitations: The PDO experiments (n=3) are underpowered, and PBMC data (Fig. 8D-E) lack sufficient donor diversity. Expanding to additional patient-derived samples (e.g., PDX models) and quantifying antigen-specific T-cell responses would enhance translational relevance.\nMinor Critiques\n4. Single-Cell RNA Sequencing\n- The reanalysis of Geller et al. (2022, Nat Commun) (Fig. 1A-E) does not clearly advance beyond the original study. The authors should clarify its novel contributions.\n- Cluster annotation (Fig. 4A-B) requires protein-level validation (e.g., Chil3+Ly6C-high macrophages).\n5. Metabolomics\n- Pathway impact analysis (Fig. 3E-F) does not adjust for multiple testing, increasing the risk of false positives.\n6. Methods Concerns\n- Fig. 1F-I: The SIINFEKL model is not CRC-relevant; justification for using ovalbumin-derived peptides is lacking.\n- Fig. 2L-N: A comparison with checkpoint inhibitors (e.g., anti-PD-1) is absent, despite their clinical relevance.\n- Fig. 7 (MC38 Model): Data on tumor-infiltrating T cells and checkpoint markers (e.g., PD-L1) are missing.\n- WPG Dosing: The β-glucan dose (1 mg/mouse) lacks human-equivalent translation, raising translational concerns.\n7. Figure Clarity\n- Tumor growth curves (Figs. 2B, 2M) should include individual data points to illustrate variability.\n- Immunofluorescence images (Figs. 5I, 8G) need scale bars and higher resolution.\n- The scRNA-seq methods (e.g., batch correction, cluster resolution) require more detail for reproducibility.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We would like to sincerely thank all of you for the evaluation of our manuscript and for providing constructive and insightful comments. The feedback has significantly strengthened our work, guiding us to improve both the experimental rigor and the clarity of presentation. The revisions and additional experiments performed in response to these comments have elevated the manuscript to a higher level, and we are deeply grateful for the time, effort, and expertise invested in this review process.\n\n[... Author responses to each reviewer point follow in the original text ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made efforts to satisfactorily address the concerns raised in the previous review.", "reviewer_2": "The authors have carefully and sufficiently addressed my questions, and I would recommend acceptance of the manuscript for publication.", "reviewer_3": "I do not think the authors have adequately addressed my previous concerns.\nFor example, last time I questioned whether FLT3 is a mediator between NK cells and DCs. Now the authors have changed FLT3 to CCL5, but provide little or no new supporting evidence. CCL5 may be less specific because: 1) NK cells are not a major producer of it in this environment, and 2) CCL5 can recruit many types of immune cells, not just DCs.\nAnother example: I questioned, “How long does trained immunity last?” The authors mentioned “several weeks” but did not cite any figure or specify the exact duration. Moreover, claiming “several weeks” may not be sufficient to support trained immunity.\nAdditionally, when I asked, “Does trained immunity induce exhaustion in immune cells, such as NK cells?” the authors only reanalyzed their scRNA-seq dataset. Why did they not assess this by flow cytometry? The authors stated they did not want to present additional data in the manuscript, but the reason for this decision is unclear.\nRegarding my concern #5, I still think the tumor-only group is important. But they authors do not provide the data.", "reviewer_4": "The authors performed new experiments, such as adoptive transfers, sequential migration assays, CD8+ T cell depletion, Seahorse assays, ChIP-qPCR, and validation in a more relevant tumor model. The manuscript now presents a compelling and well-supported narrative.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We would like to sincerely thank you for the evaluation of our manuscript and for providing constructive and insightful comments. We hope that our revisions and additional experiments performed have been adequate."}]

Decision letter