Intrahepaticcholangiocarcinoma(ICC)isanaggressivelivertumorwithlimitedtherapeuticoptionsand5-year survivalratesoflessthan10%.ICCischaracterizedbyitshighlydesmoplasticnature,withabundanceofcancer- associated fibroblasts (CAF) and extracellular matrix (ECM). The role of CAF and ECM in ICC remain controversialduetothepaucityoffunctionalinvivostudies.WhilethemajorityofICCinvitrostudiessupporta cancer-promotingroleofCAF,recentstudiesinendogenouslyarisingpancreaticcancer,ahighlydesmoplastic tumorwithmanysimilaritiestoICC,haveshownthatCAFrestraincancergrowth.Here,weseektoanswerthe question whether CAF promote or restrain ICC, using a number of novel and cell-specific tools to manipulate CAFandtumorcellsandtheircrosstalkinendogenouslyarisingICCinvivo.WehypothesizethatCAFandECM provideanichethatpromotesICCgrowthandsurvival,andthatdetailedcharacterizationofICC-CAFcrosstalk will identify novel therapeutic targets within the tumor microenvironment. In Aim 1, we will determine the role CAFusingnoveltoolstodeterminehowgeneticCAFinhibitionorearlyandlateCAFablationaffectICCgrowth, proliferativeandanti-apoptoticsignalingpathways,andmousesurvival.InAim2,wewillinvestigatepathways that mediate the recruitment, proliferation and activation of CAF in ICC focusing on the hypothesis that tumor cellshijacknormalfibrogenicmechanismintheliverviatumor-derivedTGFb? andPDGFisoformsandTGFb?- activatingintegrins,resultinginaccumulationandactivationofCAF.Inadditiontodetailedmechanisticstudies inknockoutmiceandinvitroco-cultures,wewilldeterminewhetherpharmacologicinhibitionofCAFactivation byFDA-approveddrugNintedaniborintegrin-blockingantibodiesinhibitICCgrowthandprolongmousesurvival. InAim3,wewilldeterminepathwaysthroughwhichCAFmodulateICCgrowth,focusing on the hypothesis that CAF-derived ECM activates tumor-promoting signals in the tumor cell compartment. To test this hypothesis, we will investigate ICC development in mice with CAF-specific knockout of Col1a1, or tumor-selective knockout of collagen-sensing receptor discoidin domain receptor 1 (DDR1). In addition, we will determine whether ECM- mediated stiffness and subsequent activation of mechanosensitive signaling in tumor cells promote ICC development. We will measure tumor stiffness, activation of mechanosensitive signaling pathways and DDR1 expression in human CCA samples and correlate these to clinical outcomes. The role of stiffness and DDR1 in ICC growth and CAF-ICC crosstalk will be investigated in more detail in vitro through modulation of stiffness and by co-culturing CAF and ICC cells that lack Col1a1 or DDR1, respectively. Insummary,theproposedstudies willemploynoveltoolstoansweralong-standingquestioninthefieldandmayprovideabasisfortargetingICC- CAFcrosstalkasnoveltherapeuticstrategyforthisdevastatingmalignancy.