1、Cancer CellPerspectiveObstacles Posed by the Tumor Microenvironmentto T cell Activity:A Case for Synergistic TherapiesKristin G.Anderson,1,2,3,4Ingunn M.Stromnes,1,2,4,*and Philip D.Greenberg1,2,3,*1Clinical Research Division,Fred Hutchinson Cancer Research Center,Mail Stop D3-100,P.O.Box 19024,Seat
2、tle,WA 98109,USA2Program in Immunology,Fred Hutchinson Cancer Research Center,Seattle,WA 98109,USA3Departments of Medicine/Oncology and Immunology,University of Washington School of Medicine,Seattle,WA 98109,USA4Co-first author*Correspondence:istromnefredhutch.org(I.M.S.),pgreenu.washington.edu(P.D.
3、G.)http:/dx.doi.org/10.1016/j.ccell.2017.02.008T cell dysfunction in solid tumors results from multiple mechanisms.Altered signaling pathways in tumorcells help produce a suppressive tumor microenvironment enriched for inhibitory cells,posing a majorobstacle for cancer immunity.Metabolic constraints
4、 to cell function and survival shape tumor progressionand immune cell function.In the face of persistent antigen,chronic T cell receptor signaling drives T lympho-cytes to a functionally exhausted state.Here we discuss how the tumor and its microenvironment influencesTcelltraffickingandfunctionwitha
5、focusonmelanoma,andpancreaticandovariancancer,anddiscusshowscientific advances may help overcome these hurdles.IntroductionSolid tumors commonly arise from genetic mutations in driveroncogenes and tumor suppressor genes that have profoundconsequences on cell differentiation,proliferation,survival,an
6、dgenomic stability.During cancer progression,tumor cells alternormal developmental processes to orchestrate a supportivebut overtly immunosuppressive tumor microenvironment(TME)comprised of immune cells,fibroblasts,and endothelial cells,often embedded within a robust extracellular matrix(ECM)(Ha-nah
7、an and Coussens,2012;Stromnes et al.,2014b;Whiteside,2008).From the formation of the earliest pre-invasive lesion tometastatic spread,the TME can support angiogenesis,tumorprogression,and immune evasion from T lymphocyte recogni-tion,as well as dictate response to cancer therapy.Despite thesignifica
8、nt obstacles that tumor-reactive T lymphocytes face insolid tumors,accumulating evidence indicates natural,induced,and engineered immune responses to cancer can dramaticallychange clinical outcomes,particular in certain malignancies(Chapuis et al.,2013;Galon et al.,2013;Kroemer et al.,2015;Rosenberg
9、 and Restifo,2015;Turtle et al.,2016).Such clinicalfindingssparkhopeandexcitementthatagreaterunderstandingofthe relationship between the complexcomponents ofthe TMEand immune function will inform more broadly effective immuno-therapies for intractable malignancies.Immune checkpoint blockade(e.g.,ant
10、i-PD-1,anti-PD-L1,or anti-CTLA-4),designed to amplify endogenous anti-tumorT cell responses,has revolutionized cancer treatment(Sharmaand Allison,2015).The success of this approach is notable inmelanoma and non-small-cell lung cancers that often containnumerous genetic mutations(Lawrence et al.,2013
11、),a fractionof which produce neoantigens recognizable by endogenousTcells(LuandRobbins,2016;Stronenetal.,2016).Theadoptivetransfer of genetically engineered T cells to express a receptorspecific for a tumor antigen is a more targeted approach andhas shown efficacy in melanoma(Morgan et al.,2013)as w
12、ellas tumors with lower mutational burdens.T lymphocytes engi-neered to express a chimeric antigen receptor(CAR)specificto the B cell marker CD19(Kalos et al.,2011;Turtle et al.,2016)or a T cell receptor(TCR)specific to self/tumor antigenor Wilms tumor antigen(Chapuis et al.,2013)have shown dra-mati
13、c clinical responses in hematological malignancies.How-ever,broadly translating similar approaches to treat carcinomashas proven more difficult.First,since reproducibly expressedcandidate tumor antigens are also often self-antigens,toxicitycan be limiting.Second,if tumors persist,chronic TCR signali
14、ngcan lead to a T cell-intrinsic program of exhaustion(Schietingeretal.,2016;Wherryetal.,2007).Thirdly,therearemultipleimmu-nosuppressive mechanisms operative in the TME that interferewith T cell function(Pitt et al.,2016).In addition,even if tumorcellkillingistransientlyachieved,cancerscanevadethei
15、mmunesystem by a variety of mechanisms,including outgrowth ofvariants after immunoediting(Schreiber et al.,2011).Pancreatic ductal adenocarcinoma(PDA)and high-grade se-rous ovarian cancer,which are often diagnosed at advancedstages,arelargely resistant to therapy,including immune check-point blockad
16、e(Brahmer et al.,2012;Ring et al.,2016;Royalet al.,2010).These tumors have few coding mutations,andthus contain few neoantigens,compared with melanoma andnon-small-cell lung cancer(Lawrence et al.,2013).Furthermore,while immune checkpoint blockade has yielded dramatic clinicalresponses,particularly
17、in the subset of malignancies with largemutational burdens(Hamidetal.,2013;Hodietal.,2010),clinicalresponses are often not durable(Ribas et al.,2016),indicatingthat,even in highly responsive tumors,sustaining long-lastingimmune activity is daunting.Thus,approaches that simulta-neously promote T cell
18、 anti-tumor activity and avoid/overcomethe most significant obstacle(s)in the relevant TME may provemost beneficial.Tumor cell intrinsic genetic mutations can coordinate theinduction of downstream and paracrine signaling pathwaysculminating in chronic fibroinflammatory states.These changesinfluence
19、cell composition,ECM,vasculature,nutrient availabi-lity,bioenergetics,and angiogenesis.Direct links betweenTME components and immune system suppression and evasionCancer Cell 31,March 13,2017 2017 Elsevier Inc.311are increasingly being recognized(Pitt et al.,2016).Metabolicdemands ofboth tumor cells
20、and thesupportive stromalnetworklimit nutrient availability,and concurrently overexpose T cells tosuppressive metabolites,thereby reducing T cell effector func-tion(Chang and Pearce,2016).Persistent antigen can causechronic TCR signaling and T cell exhaustion,leading to epige-netic changes that may
21、not be readily overcome by immunecheckpoint blockade(Pauken et al.,2016;Sen et al.,2016).Finally,the heterogeneity of tumors,not only between distinctmalignancies but also different individuals with the same typeofcancer,oreventumorlesionsinthesamepatient,complicatestreatment strategies(Hugo et al.,
22、2016;Makohon-Moore and Ia-cobuzio-Donahue,2016;Schwarz et al.,2015).The design ofsuccessful immunotherapies must account for mutational andantigenic heterogeneity as well as TME complexity.The diversearray of immune-evasion mechanisms employed by tumorshighlights the necessity for combination and sy
23、nergistic thera-pies.Here,we discuss major drivers of T cell dysfunction in thecontext of three solid tumors:(1)melanoma,in which T cell re-sponses exist and frequently can be enhanced to mediate anti-tumor activity(Rosenberg and Restifo,2015),(2)ovarian cancer,inwhichpotentiallybeneficialTcellrespo
24、nsesexistinasubsetofpatients,but have proven difficult to augment(Santoiemma andPowell,2015),and(3)PDA,in which endogenous T cell re-sponses are rare(Emmrich et al.,1998;Ene-Obong et al.,2013;von Bernstorff et al.,2001).Tumor Cell Intrinsic Pathways that Modulate the TMEand Interfere with ImmunityPa
25、ncreatic and ovarian cancer are each associated with severalcommon oncogenic mutations that can influence the TMEand immunity(Figure 1).Mutations in KRAS are common inFigure 1.Overview of Obstacles in the TumorMicroenvironment that Interfere with T CellTrafficking and Functionboth malignancies(Forbe
26、s et al.,2011)and play a critical role in the developmentof the TME.KrasG12Dimpacts all phasesof tumor growth:initiation,invasion,main-tenance,and metastasis in PDA(Collinset al.,2012;Ying et al.,2012).KrasG12DincreasestumorcellproliferationviaMAPK and phosphatidylinositol 3-kinase-mammalian target
27、of rapamycin(mTOR)signaling and modulates metabolism byglucose transporter 1(GLUT1)upregula-tion(Yingetal.,2012).Inadditiontoalteringintrinsic tumor cell behavior,oncogenicKras has pronounced indirect effects onneighboring cells.Introducing KrasG12Dinto normal mouse pancreatic ductal cellsinducesgra
28、nulocyte-macrophage(GM)colony-stimulating factor(CSF)expression(Pylayeva-Guptaetal.,2012),andGM-CSFproduction recruits myeloid cells that inter-fere with CD8 T cell infiltration and tumorcell killing in PDA(Bayne et al.,2012;Pylayeva-Gupta et al.,2012).GM-CSF also recruits neutrophils to the TME,whi
29、ch facil-itate PDA metastasis(Steele et al.,2016),and impair both T cellaccumulation and priming in autochthonous PDA(Stromneset al.,2014a).In addition to inducing downstream paracrinesignaling to myeloid cells,KrasG12Dincreases Hedgehog ligandsShh and Ihh expression that activate a-smooth muscle ac
30、tin-positive cancer-associated fibroblasts(CAFs)(Collins et al.,2012),leading to expansion of the fibroinflammatory stroma(Olive et al.,2009)and T lymphocyte exclusion(Feig et al.,2013;Lo et al.,2015).Finally,KrasG12Dincreases MMP7,COX2,interleukin-6(IL-6),and pSTAT3 levels(Collins et al.,2012),ther
31、eby influencing innate cell signaling,chronic inflam-mation,and ECM remodeling.Hence,a single oncogenic muta-tion can have profound effects on the development of the TMEand the anti-tumor immune response.Although active Kras mu-tants are elusive therapeutic targets,a recent study using HLA-A*11:01 t
32、ransgenic mice isolated TCRs reactive to KrasG12V7-16and to KrasG12D7-16neoepitopes,but not wild-type Kras(Wanget al.,2016),which could be useful tools for engineering T cellsfor the fraction of patients who are HLA-A*11:01+.Common pathways across distinct malignancies that maybroadly influence canc
33、er immunity are particularly intriguing.Oncogenic Kras or other signaling mechanisms can induce acti-vation of the WNT/b-catenin pathway(Lemieux et al.,2015).b-CateninisanintegralE-cadherinadaptorproteinandtranscrip-tional co-regulator,and b-catenin is commonly dysregulated inPDA(Zeng et al.,2006)an
34、d ovarian cancer(Bodnar et al.,2014)as a result of oncogenic signals or mutations in its gene(Zengetal.,2006).Activationofb-cateninisdetectedinasubsetofmel-anomasandisassociatedwithanon-inflamedtumorgenesigna-ture and low T cell infiltration(Spranger et al.,2015).b-Catenin312Cancer Cell 31,March 13,
35、2017Cancer CellPerspectivesignaling can indirectly prevent intratumoral T cell accumulationbyinhibiting production of CCL4,a chemokine critical for recruit-ment of dendritic cells(DCs),which prime naive T cells(TN)andactivate immune responses(Spranger et al.,2015).Substantialeffortsareunderwaytotarg
36、etWNT/b-cateninsignalingincancer,including the use of newly designed small-molecule inhibitors(Zhang and Hao,2015).Since these and many of the othersmall-molecule inhibitors being pursued target pathways alsofunctional in hematopoietic cells,preclinical studies in autoch-thonous tumor models in whic
37、h tumors arise in the native organand the endogenous immune and hematopoietic systems areintact should prove helpful for predicting the potential for clinicalsuccess for such approaches.Another neoplastic cell-intrinsic pathway impacting cancerimmunity is focal adhesion kinase(FAK).FAK1 and PYK2/FAK
38、2are non-receptor tyrosine kinases that regulate adhesion,invasion,migration,proliferation,and survival(Frame et al.,2010).FAK1 level is elevated in human PDA and correlates withrobust fibrosis and poor CD8 T cell accumulation(Jiang et al.,2016).Tumor cell contact with rigid ECM components,such asco
39、llagen or fibronectin,induces Rho-associated coiled-coilkinase-dependent activation of FAK1(Jiang et al.,2016).Inhibiting FAK1 or FAK2 greatly reduces cytokine production,the frequencies of CAFs,suppressive myeloid subsets,andCD4+Foxp3+regulatory T cells(Treg),as well as ECM accumula-tion.FAKinhibit
40、ionhaltstumorgrowthandincreasessurvivalinaPDA mouse model,and anti-tumor activity can be furtherimproved if combined with chemotherapy or anti-PD-1(Jianget al.,2016).FAK is implicated in suppression of anti-tumor im-munity in other cancers as well.In squamous cell carcinoma(SCC),nuclear FAK associat
41、es with key transcription factorsand induces CCL5 and transforming growth factor b(TGF-b)expression,which recruit/induce tumor Treg(Serrels et al.,2015).Pharmacological FAK inhibition causes regression ofautochthonous lung tumors in an oncogenic Kras-driven model(Konstantinidouetal.,2013).FAKcaninte
42、rferewithTCRsignalingin T cells(Chapman et al.,2013),suggesting that FAK inhibitionmay have non-redundant effects by altering both tumor andimmune cells.Furthermore,FAK signaling can induce nuclearlocalization of b-catenin and transcriptional activation of theb-catenin target gene MYC(Ridgway et al.
43、,2012),suggesting amechanistic link between these oncogenic pathways.A FAK in-hibitor in combination with PD-1 blockade and gemcitabine iscurrently being evaluated in clinical trials for pancreatic andovarian cancer(www.clinicaltrials.gov,NCT02546531).SinceT cell dysfunction in cancer is commonly re
44、stricted to the tumorsite,investigation into how such combinations alter the TMEand infiltrating T cells in patient biopsies will likely be critical foridentifying reasons for therapeutic failure and/or success.Mutations in the tumor suppressor gene TP53,which occur inmany solid tumors,are also asso
45、ciated with a suppressive TME.TP53 is mutated in approximately 96%of high-grade serousovarian cancers(The Cancer Genome Atlas Research Network,2011),75%of PDAs(Rozenblum et al.,1997),and 19%ofmelanomas(Hodis et al.,2012).In normal cells,p53 is activatedupon cell stress to induce cell-cycle arrest,DN
46、A repair,cellsenescence,and/or apoptosis.Thus,p53 loss can promotetumorigenesis by disrupting normal cell-cycle checkpoints.Several missense p53 mutants exert further oncogenic effectsby promoting tumor growth,metastasis,and modulating amore suppressive TME(Rivlin et al.,2011).p53 loss can induceove
47、rexpression of CCL2,which recruits suppressive myeloidcells into the TME(Walton et al.,2016).Mutant p53 can disruptTGF-b signaling and promote epithelial-mesenchymal transition(EMT)and metastatic invasion(Elston and Inman,2012).An EMTgene signature is associated withresistance to PD-1 blockade inmel
48、anoma and a similar EMT gene signature isprominent in PDA(Hugo et al.,2016),underscoring potentially key immunosup-pressive events linked with EMT in solid tumors.T Cell Exclusion from Solid TumorsSolid tumors can facilitate immune evasion by restricting T cellmigration.Indeed,many solid tumors are
49、classified based onthe T cell infiltrate;the presence of T cell infiltration contributesto a higher immunoscore that correlates with improved patientprognosis(Curiel et al.,2004;Galon et al.,2006).Several keyevents in lymphocyte migration contribute to T cell infiltrationincluding:(1)selectin-mediat
50、ed rolling,(2)chemokine-mediatedintegrin activation,(3)integrin-mediated arrest,and(4)extrava-sation across the endothelial barrier(Schenkel and Masopust,2014).Although many tumors overexpress vascular endothelialgrowth factor(VEGF),and rapidly growing lesions often develophypoxic regions that stimu
