Targeting the Microenvironment in Advanced Colorectal Cancer

TargetingtheMicroenvironmentinAdvancedColorectalCancer
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DanieleV.F.Tauriello1andEduardBatlle1,2
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1.InstituteforResearchinBiomedicine(IRBBarcelona).TheBarcelonaInstituteofScience
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andTechnology.BaldiriiReixac10,08028Barcelona,Spain
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2.InstitucióCatalanadeRecercaiEstudisAvançats(ICREA),Barcelona,Spain.
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Correspondence:[email protected]&[email protected]
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Keywords:stroma,cancerecology,molecularsubtypes,pre-clinicalmousemodels,
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immuneoncology,TGF-β
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Abstract
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Colorectal cancer (CRC) diagnosis often occurs at late stages when tumour cells have
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already disseminated. Current therapies are poorly effective for metastatic disease, the main
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causeofdeathinCRC.Despitemountingevidenceimplicatingthetumourmicroenvironmentin
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CRCprogressionandmetastasis,clinicalpracticeremainspredominantlyfocussedontargeting
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the epithelial compartment. As CRCs remain largely refractory to current therapies, we are
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compelled to devise alternative strategies. TGF-β has emerged as a key architect of the
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microenvironment in poor prognosis cancers. Disseminated tumour cells show a strong
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dependencyonaTGF-β-activatedstromaduringtheestablishmentandsubsequentexpansion
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of metastasis. Here, we will review and discuss the development of integrated approaches
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focusedontargetingtheecosystemofpoorprognosisCRCs.
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Trends
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In the search for new paradigms on which to base novel therapeutic strategies for
advancedCRC,focushasincreasinglybeencentredonthetumourmicroenvironment.
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Thereisanemergingnotionthatinteractionsbetweenepithelialcancercellsandtheir
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environmentcanbeunderstoodapplyingaconceptualframeworksimilartothatused
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tostudyecosystems.
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In CRC patients, a stromal-expressed gene programme enriched for TGF-β and
downstreamtargetshasbeenlinkedtopoorprognosisandmetastasisformation.
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TGF-β signalling plays key roles in instructing the tumour microenvironment of late
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stageCRCs,yetinhibitionofTGF-βsignallingasatherapeuticstrategyremainsscarcely
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exploredintheclinic.
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Thetumourepitheliumasprimarytargetofstandardtherapies
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Colorectal cancer (CRC) originates from benign lesions known as adenomas: localised
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glandular overgrowths in the epithelial lining of the large bowel. Over time, some adenomas
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accumulate mutations in signalling pathways critical to stem cell maintenance, cellular
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proliferation, and tumour suppression [1, 2] and they can evolve into invasive CRCs that may
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ultimatelyspreadtodistantorgans.Thisprocesscalledmetastasis(seeBox1)occursinabout
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40-50%ofpatientsandconfersalowprobabilityofsurvival.
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In the clinic, patients are classified in 4 stages (see Glossary). Treatment involves
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aggressive surgical resection of the primary tumour, which cures a large proportion of early
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stage patients. However, some stage II and many stage III patients will relapse, frequently in
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theformofmetastasis,asaconsequenceoftumourcellsthatdisseminatedbeforeresection.
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Unliketheprimarytumour,metastasesarelessfrequentlyremovedbysurgeryunlesslimitedin
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number and extent [3]. Therefore patients that present with metastases at the time of
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diagnosis(stageIV)andthoseatperceivedriskofrelapsereceivecytotoxicchemotherapy:in
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most cases a combination of folinic acid, 5-fluorouracil, and oxaliplatin or ironotecan. This
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strategyaimstokillhighlyproliferativecancercellsandhasbeenastapleinthetreatmentof
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solidcancersfordecades[4].AlthoughadjuvantchemotherapyisbeneficialtostageIIIpatients
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and has modest potential for improved survival in stage II [5, 6], it performs poorly in the
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metastaticsetting,almostinvariablygivingrisetodrugresistanceanddiseaseprogression.
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In second-line treatment, chemotherapy is increasingly combined with targeted therapy,
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designed to intervene with specific signalling pathways or cellular mechanisms [4]. A key
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exampleinCRCistheuseofantibodiestargetingtheepidermalgrowthfactor(EGF)receptor,
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often exploited by cancer cells to stimulate proliferation. Apart from eventual resistance, the
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mainproblemwithtargetedtherapiesisthevariableresponsesinpatients,requiringabetter
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griponpredictivebiomarkers[7].Forinstance,mutationsthatactivatetheMAPK/ERKpathway
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downstream of EGFR, such as those frequently occurring in the KRAS and NRAS GTPases or
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BRAF,canrenderanti-EGFRtherapyineffective[8].
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A better understanding of advanced cancer may lead to more effective therapies for
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metastaticCRC,butisalsohighlyrelevanttoimprovethemanagementofearlierstagesofthe
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disease.ArguablythemostimportantquestionforstageI-IIIpatientsiswhetherornottotreat
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at all and which therapeutic strategy will be beneficial to prevent recurrence. Assessment of
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probability of relapse after therapy on the individual level remains a major challenge [9].
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Anatomicalandhistopathologicalfeaturesofthetumoursuchasextentofinvasion(T),number
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of lymph node metastases (N), bowel perforation or obstruction, the presence of
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lymphovascularinvasion,andapoorlydifferentiatedhistologyareusedtoidentifyCRCpatients
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atriskofrecurrence.However,theseparametersonlyholdamoderatepredictivepowerand
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donothelpselectoptimaltreatmentoptions.Furthermore,withtheexceptionofchromosomal
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or microsatellite instability (MSI) [10, 11] and mutations in the BRAF oncogene [12], no
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molecularfeatureisrobustlyassociatedwithprognosisandthereforeusedroutinelyinclinical
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practice[9,13].
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TranscriptomicsredefiningCRCclassification
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Toimprovepatientstratificationandidentifypotentialmoleculartargetsfortherapy,the
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field has generated large collections of transcriptomic datasets from tumour samples, which
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have enabled the identification of CRC subtypes based on distinctive global gene expression
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profiles[14-19].Inanattempttoconsolidatethesedata,arecentmeta-analysisdividedCRCs
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into 4 defined consensus molecular subtypes (CMS), representing an MSI-like class (CMS1), a
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canonical WNT/MYC class (CMS2), a metabolically dysregulated class (CMS3), and a
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mesenchymalclass(CMS4)[20].Thelattersubtype,towhichabout1inevery4CRCsbelongs,is
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ofparticularinterestasitrelapseswithhigherfrequencythantheothers.
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Theelevatedexpressionofmesenchymalgenesinanepithelialcancerledtotheproposal
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that epithelial-to-mesenchymal transition (EMT) characterizes poor prognosis in CRC [21].
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However,thetranscriptomeofatumoursamplenotonlyreflectstheexpressionprogrammeof
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epithelial cancer cells but also the profile of mesenchymal cells present in the tumour
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microenvironment(TME).Indeed,ourgroupandthegroupofEnzoMedicorecentlydiscovered
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thatexpressionofmesenchymalgenesintranscriptomicCRCdataislargelycontributedbycells
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oftheTME,mainlybycancer-associatedfibroblasts(CAFs),ratherthanbycancercells[22,23].
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In agreement with these observations, CMS4 cancers show a higher degree of stromal
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infiltration than the other subtypes [20]. Furthermore, our analyses indicate that a large
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proportion of these CAF-expressed genes are strongly associated to cancer relapse and poor
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prognosisinCRCcohorts[22,23].Ofnote,thefactthattheexpressionofmesenchymalgenes,
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includingmanyEMTmasterregulatorssuchSNAI1,TWISTandZEB1,iscontributedbystromal
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cellsintranscriptomicprofilesdoesnotruleoutthepossibilitythatsubsetsoftumourcellsmay
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undergoEMT,particularlyatinvasionfronts.
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TreatingCancerasanEcosystem
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Focussing on the tumour stroma is not novel. At the end of the 19th century, English
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physician Stephen Paget – observing preferential patterns of metastatic dissemination to
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particularorgans–proposedthe‘seedandsoil’hypothesistoexplainthisphenomenonasthe
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combination of the colonizing cancer cells’ adaptability and a congenial microenvironment in
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thedistantorgan[24].HesuggesteditwouldbeusefultostudythepropertiesoftheTME,as
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wellasitscrosstalkwithcancercells.Indeed,theroleoftheTMEbothattheoriginalsiteandin
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thedistantorganhasbecomeatopicofintenseinvestigationinrecentdecades[25].Similarto
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howintestinalcryptstemcellsareregulatedbyaspecializedstemcellmicroenvironmentcalled
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aniche,theTMEcanfostercancerstemcellsandthusdriverecurrenceandmetastasis(Box1)
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[26,27],aswellasprovidemechanismsfordrugresistance[28,29].Itisnowacceptedthatthe
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TMEisanactiveplayerintumourmalignizationandthatitprovidesafertilegroundtodelvefor
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therapeutictargets[30,31].
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The study of complex interactions between cancer cells and their environment inspires
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parallelswiththeinterrelationshipsrecognizedinanecosystem.Theapplicationofecological
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conceptstooncologymayhelpexplainwhyarigidfocusonepithelialcancercellsalonehasled
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toahighnumberoffailednewdrugsandclinicalstrategies.Justasecologyistheframeworkof
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choice for understanding the mechanisms of organismal evolution through natural selection,
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ecology working on the level of cellular populations serves to clarify the dynamics of cancer
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evolution. This includes natural selection due to limited resources as well as the artificial
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selectionofsubclonesresistanttotherapy.Hence,thefitnessofacellularpopulation(orofa
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set of cancer mutations) should be considered in the context of its local environment, which
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consistsofcellularinteractions,physicalparameters(oxygenlevels,pH,mechanicalpressure),
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andmoleculeslikeextracellularmatrixcomponents,growthfactorsandcytokines.
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Moreover,ecologicaltypesofinteractioncandescribevariouslevelsofcrosstalkintumour
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dynamics. Examples of competitive and cooperative interactions have been reported both
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betweendifferentepithelialsubclonesandbetweenepithelialcancercellsandthestroma[32-
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34].Additionally,theimmunesystemcanbeconvertedfromapredatortoanaccomplice(see
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Box2).Indeed,thestromalenvironmentisnotstaticandco-evolutionisacompellingconcept:
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parallel evolution of neoplastic epithelial cancer cells and cells in the microenvironment,
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subject to epigenetic and gene expression changes [35]. Furthermore, the stromal
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compartment contributes significantly to intratumoral heterogeneity, with important
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implications for disease progression and therapeutic responses [36]. Thus, cancer ecology
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integrates complex interactions between epithelial cancer cell populations and their
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environmenttopromiseadeeperunderstandingofthebiologyofcancer[37,38].
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ModulatingtheCRCecosystemfortherapeuticpurposes
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The composition of the TME during colorectal tumorigenesis and in advanced cancers is
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subject to intensifying inquiry (for a recent overview see refs: [39, 40]) and is found to be
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different from normal intestinal stroma (see Figure 1, Key Figure). As recently reported, the
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majorityofgenesthatpredictcancerrecurrenceinCRCpatientsareexpressedinCAFsandnot
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bythecancercells[22,41].ThestrikingprognosticpoweroftheTMEraisestheopportunityto
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more accurately estimate the risk of relapse of any given stage I-III patient. It also paves the
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wayforthedevelopmentofadditionallyrefinedmolecularclassificationsbasedonthecontents
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of the ecosystem. A relevant early example of such an ecological classification of CRC was
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establishedbyGalonandothers[42-44],whoobservedthatinfiltrationofparticularsubsetsof
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Tcellsinthetumourmasspredictslongerdiseasefreesurvivalintervalsaftertherapy.
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Moreover,novelCRCclassificationsbasedonthetypeandfeaturesofstromalcellswillbe
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key to stratify patients for treatments that target the TME. Based on the idea that CRC cells
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dependonparticularstromalfactorstoeffectivelydisseminate,therapeuticsthatmodulatethe
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CRCecosystemmaybeeffectiveintreatingorpreventingmetastasis.Anadditionaladvantage
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of targeting the TME is its genetic stability, making drug resistance less likely to occur.
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Furthermore,thecommonalityofmostelementsinthestromaacrosscancertypessuggestsa
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broad applicability of effective therapies. A handful of therapies based on the above concept
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have already been developed and are currently being applied. For instance, blood vessel
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formation and endothelial cells have been successfully targeted by anti-angiogenic therapy
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[45]. Indeed, a monoclonal antibody against vascular endothelial growth factor (VEGF, i.e.
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bevacizumab) inhibits angiogenesis and has been shown to improve survival for stage IV
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patients[46,47].Additionally,metronomicchemotherapy–aimingtoforestalldrugresistance
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and suppress neo-angiogenesis – has shown promise for prolonging survival and limiting
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metastasisinpreclinicalmodels,andisbeingtestedinclinicaltrials[48,49].
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AnotherprimeexampleoftherapydirectedtotheTMEisimmunotherapy(Box2).Ithas
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beenproposedthatseveral(chemo-)therapiesthatdonotdirectlytargetimmunity,suchas5-
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fluorouracil and platinum compounds, in fact do rely on immune components. These
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conventional treatments are effective at least in part by reinforcing tumour
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immunosurveillance,especiallywhentheyinduceimmunogeniccancercelldeath[50].
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TGF-βasamainorganizerofthemetastaticCRCecosystem
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TGF-β has a dual role in CRC, with tumour suppressive functions in early stages and
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promotingdiseaseprogressioninadvanceddisease(seeBox3).Ourgroupdemonstratedthat
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elevatedTGF-βsignallingintheTMElinkstopoorprognosisinCRC[22,41].ThestromalTGF-β
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responseencodesageneprogrammethatincludesaplethoraofcytokines,growthfactorsand
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extracellularmatrixproteins,manyofwhichhavebeenshowntoplaykeyrolesduringdisease
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progressionandmetastasisinseveralcancertypes.Amongthesepotentialtherapeutictargets
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isinterleukin-11(IL-11),acytokinesecretedbyTGF-β-stimulatedfibroblaststhatinducespro-
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survival response in CRC cells during cancer progression and metastatic colonization [41, 51].
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The picture that emerges is that key functions required to complete the formation of
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metastasisareprovidedbytheTGF-β-activatedmicroenvironment(Figure1).
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Because disseminated tumour cells thrive on a TGF-β-activated environment, this
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dependencycouldbeexploitedintheclinicalsettingtoimprovepatienttreatment;ourgroup
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showedforthefirsttimethatpharmacologicalinhibitionofstromalTGF-βsignallingiseffective
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in blocking metastatic colonization in mice [22, 41]. Besides activating a pro-metastatic
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programme in CAFs [52], stromal TGF-β signalling induces a pro-tumorigenic phenotype in
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neutrophils and macrophages [53-55]. In addition, TGF-β directly suppresses the anticancer
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response of the adaptive immune system, including the deregulation of cytotoxic T
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lymphocytes[56-58].Also,neutralizingtheTGF-βpathwayinTcellsledtoimmune-mediated
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eradicationoftumoursinamodelofmelanoma[59].ThatthedualroleofTGF-βsignallingin
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instructing immune cells and CAFs might be more related than is immediately obvious, is
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suggestedbyreportinwhichCAFswereshowntohaveimmunesuppressiveeffects[60,61].
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Thus, it will be interesting to assess the efficacy of pharmacological intervention in this
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pathway on the CRC ecosystem. Several therapeutic strategies targeting TGF-β are in clinical
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development (see Box 4) [62-64]. This spurs the hope that if we can block TGF-β
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pharmacologically, we may prevent a vicious cycle of stroma activation, expression of pro-
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tumorigenic secreted factors, and the generation of an immunosuppressive environment
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(Figure 1). Indeed, TGF-β inhibition may work as (or synergize with other types of)
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immunotherapy(Box2).
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CaveatsaboutthedualroleofTGF-βsignallinginCRC
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DuetothetumoursuppressivefunctionofTGF-βsignallinginepithelialcomponentofCRC
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(see Box 3), elevated TGF-β levels necessary to instruct the poor prognosis-associated
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microenvironmentmightnotbecompatiblewithtumourgrowth.Wespeculatethatthelossof
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TGF-βresponseintheepithelialcomponentofthecancermayfacilitatetheelevationofTGF-β
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levels during tumour progression. A functional link between these two events has been
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observedinexperimentalmodels [65].InabiobankofCRCorganoids,independenceofTGF-β-
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mediatedgrowthinhibitionwasassociatedwithadvancedstagesofthedisease[66],whereas
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lossofSMAD4inepithelialCRCcellscorrelatedwithpoorprognosisinseveralstudies[67,68].
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Yet,abrogationofthecytostaticTGF-βresponseinCRCcellsisnotexclusivelyexplainedbyloss-
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of-function mutation in pathway components suggesting additional mechanisms of TGF-β
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resistance [66]. Also, a proportion of TGF-β-reactive CRCs may respond to the cytokine by
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undergoing EMT while bypassing the cytostatic effect. Apparently, this effect is relevant to
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trigger invasion of a particular subset of early lesions named sessile serrated adenomas [69],
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whichmightthereforebenefitfromanti-TGF-βtherapy.Conversely,itremainsunclearwhether
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anti-TGF-β therapy would be safe for patients with cancer cells that have an intact TGF-β
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pathway–eveniftheyrepresentasubclone–andarekeptincheckbyitscytostaticeffects.
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Although the TGF-β pathway has emerged as a powerful architect of the pro-metastatic
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poorprognosisTME,itseffectonindividualstromalcelltypesispleiotropicandincompletely
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charted. Accordingly, we propose that, to treat the cancer ecosystem with more integrated
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approaches, we need to study and understand its complexity in greater detail. As such,
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manipulatingamasterregulatorlikeTGF-βinsufficientlycomplexmodelsystemsofmetastatic
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CRCshouldnotonlyprovidetheneededrationaleforclinicaltranslation,butwouldadditionally
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giveusrelevantparameterstohelpmapthecrosstalkinthecancerecosystem.
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Needforbetterpreclinicalmodelsmovingforward
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To study the efficacy of above-mentioned and other stroma-directed therapies in the
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preclinicalsetting,thereisademandformorecomprehensive,predictivemodels.Althoughthe
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fieldhasmovedfromsubcutaneouslyinjecting2D-culturedhumanCRCcelllinestoexploiting
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3Dorganoidcultureandpatient-derivedxenografts(PDX)–approachesthatpromisetoretain
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tumour heterogeneity in genetics, architecture and drug responses [70-72] – these in vivo
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models lack an intact TME. This is due to a mismatch between some murine and human
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signallingmoleculesandbecauseoftheabsentorabrogatedimmunesystemrequiredforthe
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engraftment of human tumour tissue in murine hosts. To address these problems, mouse
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modelsarebeinggeneratedthatcorrectknownsignallingdeficitsaswellasfeaturehumanized
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immune system components, potentially leading to patient-specific immune environments.
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Theseadvancespromisetostronglyenhancecurrentmodelsforcancerbiologyandpreclinical,
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personalizedoncology.
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An alternative approach to study the CRC TME and cancer immunity involves genetically
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modified mouse models (GEMMs), which feature an intact, immunocompetent TME yet
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typicallyfailtocaptureadvancedCRC.Thisleavesagap:totestcurrentideasanddiscoverand
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develop novel therapeutic strategies we are in need of CRC models that are humanlike in
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molecular characteristics, genetics and histopathology, and that are metastatic in a fully
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immunocompetentenvironment.Theidealscenariowouldincludeatransplantable(organoid)
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system in the commonly used C57BL/6 strain, so that it can be leveraged across a wealth of
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existinggeneticmodelstodissectspecificfunctionsofstromalpathways.Furthermore,sucha
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systemwouldbecriticaltotestingnoveltherapiesthattargetthetumourecosystem.
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Concludingremarks
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The CRC oncology field has evolved in the past decades. From histological analyses to
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molecularsubtyping,fromcytotoxicchemotherapytopersonalizedcombinationswithtargeted
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therapy.Whathasnotchanged,unfortunately,isthatbesidessurgerythereisverylittlewecan
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dotoimproveoverallsurvivalforadvanceddisease.
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However, the paradigm shift that points to the TME as a critical factor in determining
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metastatic success opens the door to a more integrated understanding of the biology of
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metastasis. One that invites us to consider cancer as a complex ecosystem rather than as a
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tenacious weed. In this context, therapeutic targeting of stromal TGF-signalling might be
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expectedtoreprogrammethepoorprognosiscancerecosystem(Figure1),withthepotential
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to make metastatic lesions unsustainable or to prevent metastatic initiation altogether. We
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hopethatthisemergingconceptwillfindatimelytranslationtotheclinic.Acknowledgingthe
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many challenges ahead (see also Outstanding Questions), we need more sophisticated and
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predictive CRC models to show proof-of-principle results that may catalyse the effort of
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targetingtheTME.
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OutstandingQuestions
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• WhattriggerstheupregulationofTGF-βinsometumoursbutnotothers?
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• What cell type is (or which cell types are) the source of secreted and activated TGF-β
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thatreprogramstheCRCecosystem?
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• As not all CRCs have known mutations in the TGF-β pathway, and some tumours may
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haveco-existingmutantandsensitivesubclones,towhatextentistheresponsivenessof
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epithelial cells to TGF-β (mainly associated to early disease stages) still relevant in
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advancedCRC?
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• Andwithinthiscontext,isTGF-βtherapysafeforallpatients?
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• Given the dependency of metastatic initiation on a TGF-β-activated TME, is the TGF-β
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therapeuticwindowlimitedtoadjuvanttherapy–toinhibitmetastaticcolonization–or
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couldTGF-βtherapyalsotreatestablishedmetastases,e.g.asimmunotherapy?
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Box1.CRCmetastasis:biologyandtherapeuticscope
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Initial transformation of the colon epithelium and subsequent transitions through the
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adenoma-carcinoma sequence are thought to require successive genetic alterations in 4
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signalling pathways: WNT, EGFR, TGF-β and P53 [1, 2]. In the healthy colonic mucosa, these
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signallingpathwaysregulatethebehaviourofintestinalstemcells(ISCs).Therefore,duringCRC
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progression,tumourcellsbecomeindependentfromthesecryptnichesignals[66].About40%
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of all CRC disseminate to other organs. Metastasis can either be present during diagnosis or
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reveal itself months or years after surgery as a recurrence. In many cases, distant recurrence
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targets the liver, at least in part because intestinal blood vessels drain into the portal vein,
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whichconnectstotheliver.Metastasisisamultistepprocessthatrequirescellstodetachfrom
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their epithelial neighbours, invade the submucosa, intravasate and survive in the vasculature
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(bloodorlymphaticvessels),extravasateandsurviveinanalienorgan,toeventuallyreinitiatea
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thrivingtumour[73,74].Thereismuchofthebiologyofmetastasisthatwedon’tunderstand,
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butfromwhatweknow,itisaveryharshandinefficientprocess[75,76].Arguably,theprocess
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selectsforthefiercestandmostresilientcancer(stem)cellsanddiscoveringwhatmakesthem
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successfulmightuncovertherapeutictargets.Importantly,themetastaticprocesshasnotbeen
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linked to specific genetic alterations within epithelial CRC cells [77]. Therefore, studies have
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shiftedfocusontotheroleoftheTMEandthemechanismbywhichthemetastaticcellexploits
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the TME for survival, immune evasion, and growth stimuli [78, 79]. Perhaps, success in
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metastasisisdefinedaswhocanbestremodeltheirmicroenvironment.
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Box2.Cancerinflammation,immunityandimmunotherapy
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Inflammation is a well-described risk factor in gastrointestinal tumorigenesis [80], and
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encompassesdisparatesignallingpathwaysbyavarietyofimmunecellsthatimpacteverystep
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of cancer progression and metastasis [81]. Although the immune system can be a powerful
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tumour suppressor by the coordinated elimination of aberrant cells, tumours find a way to
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surviveinspiteofimmunosurveillance.Thereisevidenceforimmunoselectioninmanycancers,
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includingCRC–wherethenumberofobservedneoantigens(inremainingsubclones)islower
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thanexpectedbasedonmutationrates–andcancercellscanacquireresistancetoimmunity
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bye.g.abrogatingantigenpresentation[82,83].
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Moreover,ithasbecomeincreasinglyclearthatinflammatorymechanismsintumourscan
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alsohelpdrivecancerprogression[81].Bystudyinghowprogressingcancerssuppresstheanti-
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tumour response and subvert immune regulation to foster immune suppressor cells and
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produce pro-tumorigenic factors that support cancer cells, the expansive field of cancer
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immunology has introduced a multitude of novel therapeutic strategies [84, 85]. On the
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conceptuallevel,manysuchtherapiesaimtoenhanceendogenousanticancerimmunitythatis
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somehowinsufficientordysfunctional[86].Toachievethiseffect,atherapycanreducecellular
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or molecular crosstalk mechanisms of immune suppression and/or increase T cell survival,
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proliferation,infiltration,andactivationintheTME[87].
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The promise of immuno-oncology has recently been demonstrated by long-lasting
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responsestoimmunecheckpointinhibitors.Theseprovedbeneficialinvarioustypesofcancer
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by unleashing T cells that were crippled by signalling either from immune suppressor cells or
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cancer cells hijacking anti-inflammatory mechanisms [88, 89]. It is still unclear whether these
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therapieswillbroadlybenefitCRCpatients.However,onestudyonmismatchrepair-deficient
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(MSI) cancers, including of the colon, did show therapeutic responses [90]. This supports the
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concept that tumour cells with higher frequency of neoantigens, such as in microsatellite
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instable cancers, are generally more vulnerable to T cell-mediated anti-tumour immunity,
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possiblyexplainingtheirrelativelygoodprognosis[91,92].Ifcurrentimmunotherapiesturnout
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nottohaveabroadapplicabilityassingleagentsinCRC,thereisstillhopethatacombination
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of treatments that both enhances intratumoral immune infiltration as well as activation will
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farebetter[93,94].
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Box3.Thedual,biphasicroleofTGF-βinCRC
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Thepro-metastaticeffectsofTGF-βsignallingonthetumourmicroenvironmentcanoccur
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independently of signalling in epithelial cancer cells, as many cancers silence the epithelial
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pathwaysoastoprogress.TGF-βsignallingisconsideredatumoursuppressorpathwayincolon
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carcinogenesisbecauseittriggersapotentcytostaticresponseinepithelialcells[22,65,95-97].
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Indeed, about 40% of CRCs display loss-of-function mutations in TGF-β pathway components,
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which are acquired around the adenoma carcinoma transition [2, 98]. An early clue for a
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positive role for TGF-β signalling in disease progression was found by Kawata and colleagues
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whoreportedthatserumlevelsofTGF-β1predictrelapseinCRCpatients[99].Later,ourgroup
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showedthatTGFB1,TGFB2andTGFB3mRNAlevelsassociatewithshorterdiseasefreesurvival
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intervalsaftertherapyinstageI-IIIpatients[41].ElevatedTGFB1-3expressioncorrelateswith
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upregulationofTGF-βresponsegenesignatures(TBRS)incellsoftheTME,specificallyinTcells,
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macrophages, endothelial cells and most prominently CAFs. These TBRSs hold a striking
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predictivepowerforcancerrecurrenceinCRC[41]anddetectionbyimmunohistochemistryof
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severalproteinsupregulatedinTGF-β-activatedCAFsidentifiespatientsathighriskofrelapse
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[22].Furthermore,comparisonofthemolecularsubtypesrevealedthatthemainsignallingaxis
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deregulated in the stromal/mesenchymal subtype (CMS4) is the TGF-β pathway [20]. A
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significant proportion of the genes differentially expressed in CMS4 cancers that predict a
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higherprobabilityofrecurrenceareincludedintheTBRSsandcorrespondtogenesinducedby
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TGF-β in CAFs. The role of a TGF-β-activated ecosystem in disease progression has been
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confirmed in experimental models of advanced CRC, where enforced TGF-β signalling in the
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TMEstronglyincreasesmetastaticburden[41].
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Box4.TGF-βtherapeuticsinclinicaldevelopment
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Several methods of targeting the TGF-β pathway have been described. These include
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antisense oligonucleotides to TGF-β ligand mRNA, small molecule inhibitors of the receptor
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kinasedomains,andmonoclonalantibodiesagainstligandsorreceptors.Inaddition,thereare
15
vaccines and adoptive cell transfer strategies that target TGF-β signalling as part of their
16
mechanism. For an overview of all (pre-) clinical strategies, see references [62-64]. Here, we
17
selectedTGF-βtargetingtherapiesthatarecurrentlyinclinicaltrialsforCRC(TableI).
18
19
20
17
1
TableI.TGF-βtargetedtherapiesinclinicaltrialsforCRC
Type
Drug
MoA
Antisenseoligos
Trebedersen
ASO to TGF-β2
(AP12009 Antisense mRNA
Pharma)
Receptorinhibitors
Kinase
Galunisertib
TβRI(ALK5)kinase
inhibitors
(LY2157299EliLilly) inhibitor
MAbs
TEW-7197,
MedPactor
IMC-TR1
(LY3022859EliLilly)
PF-03446962
(Pfizer)
Setting(Phase)
TrialIdentifier
Melanoma,
pancreas,CRC(I)
NCT00844064
Rectalcancer(II)
Study
cancer
immunology in solid
tumours(I)
Checkpoint inhibitor
combination in solid
tumours(I/II)
TβRI(ALK5)kinase Solidtumours(I)
inhibitor
TβRII neutralizing Solidtumours(I)
MAb
ALK-1 neutralizing Metastatic CRC (I)
MAb
Solidtumours(I)
NCT02688712
NTT02304419
NCT02423343
NCT02160106
NCT01646203
NCT02116894
NCT00557856
2
3
4
5
Immunotherapy
Vaccines
FANG
vaccine shRNA
against MetastaticCRC(II)
NCT01505166
(Gradalis)
furin*
TAGvaccine
TGF-β2-antisense/ Metastatic
solid NCT00684294
GMCSF modified tumours(I)
autologous
tumourcells
MoA:Modeofaction.ASO:antisenseoligo.ALK1:Activinreceptor-likekinase1(non-canonical
TGF-βreceptortypeIinendothelialcells).*AspartofitsMoA,autologoustumourcellscarry
bifunctionalshRNAblockingfurin,leadingtolowerTGF-β1andTGF-β2levels.
6
7
18
1
2
3
Glossary
Adjuvantchemotherapy:chemotherapygivenafterprimarytreatment(surgeryinCRC)to
lowertheriskofthecancercomingback.
4
Cancer evolution: the theory that within a tumour, accumulating mutations and/or
5
localizedselectivepressureleadstothegenerationofsubclonesthatcompetewitheachother
6
forlimitedresources.Furthermore,atthetimeofdiagnosisa(minority)subclonemayalready
7
carryamutationthatrendersit(partially)resistanttochemotherapy.Boththisconceptandthe
8
realization that this theory helps explain the unique nature of each individual tumour make
9
cancerevolutionahighlyrelevantconceptthatformsthebasisformoreintegrated(ecological)
10
therapeuticstrategies.
11
CRC staging: The American Joint Committee on Cancer (AJCC) has established a general
12
cancerstagingprotocolcalledtheTNMStagingSystem.Tstandsfortumourandevaluatesthe
13
original (primary) tumour in grades of aggressiveness; N stands for lymph Node involvement
14
andqualifiesthepresenceofcancercellsspreadingtonearbylymphnodes.Maddressesthe
15
presence or absence of distant metastasis (spreading of cancer to distant organs). Based on
16
these3categories,patientsaregroupedintofourstagesindicatedbyRomannumerals(I,II,III
17
and IV). While statistically representing significantly different risk groups for recurrence and
18
cancer-relateddeath,thestagingsystemdoesnotaccuratelypredictonthelevelofindividual
19
patients.
20
21
Cytokines: large class of small secreted signalling proteins involved in cellular crosstalk,
especiallyrelevantfortheorchestrationofimmuneresponsesandcancer.
22
Ecology: the comprehensive study of the distribution, abundance and dynamics of
23
organisms, their interactions with others and with their physical environment. It is most
24
frequently applied on the organism level, where populations and their environment form an
25
ecosystemandthelargestsuchsystemstudiedistheecosphereearth,yetitcanalsobeapplied
26
to cells in a tissue or in a cancer. Central concepts in ecology include cooperation and
27
competition,parasitismandpredation,andevolution.
28
Immunosurveillance:theprocessbywhichmalignantcellsarekeptincheckbyanticancer
29
immunity. Cancer immunity is understood to have an initial elimination phase before the
19
1
balance is tipped and tumour cells start escaping from immunosurveillance. Between
2
eliminationandescape,theremaybeaprolongedstateofequilibriumbetweencancergrowth
3
andimmunity-relatedkilling.
4
Relapse/Recurrence:thereturnofatumouraftersurgicalremovaloftheoriginaltumour
5
(whichoftenentailedremovalofpartsorthewholeofthecolon).Thetumourcanreappear,
6
oftenasadistantmetastasis.
7
Metronomicchemotherapy:frequentorcontinuousadministrationoflownon-toxicdoses
8
with no interruptions for long periods to prevent resistance and target both epithelial cancer
9
cellsandthegrowingtumourvasculature.
10
MicroenvironmentorStroma:supportiveconnectivetissuewithstructuralandfunctional
11
roles both during homeostasis and in disruption such as wounding or disease. The stroma
12
includesfibroblasts,bloodandlymphaticvessels,immunecells,andtheextracellularmatrix.In
13
thecontextofcancer,thestromaisincreasinglyunderstoodasacomplex,dynamicentitythat
14
istransformedbyandco-evolveswithcancercellsandcandrivemalignantprogression.
15
Microsatellite instability (MSI): due to a deficiency in DNA mismatch-repair genes, MSI
16
patients accumulate spontaneous errors in regions of their genome that are repetitive and
17
therebychallengingtoreplicate.
18
20
1
Figurelegend
2
Figure 1 (Key Figure). The CRC ecosystem and the progression-driving role of TGF-β.
3
Besides fibroblasts (CAF), endothelial cells, pericytes and mesenchymal stem cells (MSC),
4
colorectal cancers are infiltrated by a variety of innate and adaptive immune cells, including
5
lymphocytes (T cells, B cells, natural killer cells), monocytes, macrophages, dendritic cells,
6
granulocytes (neutrophils, basophils, eosinophils, and mast cells), and myeloid-derived
7
suppressorcells(MDSC).
8
TGF-β has emerged as a central architect that drives malignization of the cancer ecosystem,
9
activating stromal cells towards pro-tumorigenic differentiation, inducing the expression of
10
secretedfactors,andfacilitatingtheaccumulationofimmunesuppressiveanddrugresistance
11
functionalities.EffectiveecologicaltreatmentmightincludeacombinationofTGF-βinhibition
12
withotherstroma-directedtherapies,suchaschemotherapyandtargeted(immuno-)therapy.
13
14
Acknowledgements
15
Workintheauthors’labhasbeensupportedbygrantSAF2014-53784_RandaJuandela
16
Cierva fellowship (D.V.F.T) from the Spanish Ministry of Economy and Competitiveness
17
(MINECO),andbytheDr.JosefSteinerFoundation.IRBBarcelonaistherecipientofaSevero
18
OchoaAwardofExcellencefromtheMINECO.WewouldliketothankElenaSanchoforcritical
19
readingofthismanuscript,aswellastheBatllelabforfruitfuldiscussions.
20
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26
Cancer activates Stroma
Immunotherapy
TGF-β inhibition
Stroma secretes
Protumorigenic factors
TME drives Immune
Evasion & Drug Resistance
Stromal therapy
Epithelial
Cancer cell
CAF
MDSC
Dendritic Cell
Monocyte
Macrophage
Endothelial cell
Pericyte
MSC
Lymphoctes
Granulocyte
Extracellular
Matrix