1、ArticleTumorExosomalRNAsPromoteLungPre-metastaticNiche Formation by Activating Alveolar EpithelialTLR3 to Recruit NeutrophilsGraphical AbstractHighlightsdTLR3 deficiency reduces lung metastasis in the spontaneouscancer metastatic modelsdHost TLR3 promotes lung pre-metastatic niche formation vianeutr
2、ophil recruitmentdTumor exosomal RNAs activate alveolar epithelial TLR3 toinduce chemokinesdHigh TLR3 level and neutrophil infiltration in lung cancerpredict poor prognosisAuthorsYanfang Liu,Yan Gu,Yanmei Han,.,Xiaoqing Xu,Jianming Zheng,Xuetao CaoCorrespondencecaoxtimmunol.orgIn BriefLiu et al.demo
3、nstrate that TLR3 in hostalveolar epithelial cells is critical forneutrophil recruitment and lung pre-metastatic niche formation.Mechanistically,small nuclear RNAs fromprimary tumor-derived exosomesactivate TLR3,which leads to chemokinesecretion and neutrophil infiltration.Accession NumbersGSE76397G
4、SE80678Liu et al.,2016,Cancer Cell 30,243256August 8,2016 2016 Elsevier Inc.http:/dx.doi.org/10.1016/j.ccell.2016.06.021Cancer CellArticleTumor Exosomal RNAs Promote Lung Pre-metastaticNiche Formation by Activating Alveolar EpithelialTLR3 to Recruit NeutrophilsYanfang Liu,1,4Yan Gu,1,4Yanmei Han,1Qi
5、an Zhang,1Zhengping Jiang,1Xiang Zhang,1Bo Huang,2Xiaoqing Xu,2Jianming Zheng,3and Xuetao Cao1,2,*1National Key Laboratory of Medical Immunology,Institute of Immunology,Second Military Medical University,800 Xiangyin Road,Shanghai 200433,China2National Key Laboratory of Medical Molecular Biology,Dep
6、artment of Immunology,Institute of Basic Medical Sciences,Peking Union Medical College,Chinese Academy of Medical Sciences,Beijing 100005,China3Department of Pathology,Changhai Hospital,Second Military Medical University,Shanghai 200433,China4Co-first author*Correspondence:caoxtimmunol.orghttp:/dx.d
7、oi.org/10.1016/j.ccell.2016.06.021SUMMARYThe pre-metastatic niche educated by primary tumor-derived elements contributes to cancer metastasis.However,the role of host stromal cells in metastatic niche formation and organ-specific metastatic tropismis not clearly defined.Here,we demonstrate that lung
8、 epithelial cells are critical for initiating neutrophilrecruitment and lung metastatic niche formation by sensing tumor exosomal RNAs via Toll-like receptor3(TLR3).TLR3-deficient mice show reduced lung metastasis in the spontaneous metastatic models.Mechanistically,primary tumor-derived exosomal RN
9、As,which are enriched in small nuclear RNAs,activateTLR3 in lung epithelial cells,consequently inducing chemokine secretion in the lung and promoting neutro-phil recruitment.Identification of metastatic axis of tumor exosomal RNAs and host lung epithelial cell TLR3activation provides potential targe
10、ts to control cancer metastasis to the lung.INTRODUCTIONTumor metastasis is the leading cause of cancer-related mor-tality,which involves dissemination of cancer cells to distant or-gan sites and their adaptation to foreign environments(Hana-han and Weinberg,2011).Each of these processes is drivenby
11、 cooperation between tumors and their microenvironments.The primary tumor has been proposed to be able to educatethe secondary sites by promoting the formation of supportivemetastatic environments,termed the pre-metastatic niche(Ka-plan et al.,2005).In detail,bone marrow-derived cells(BMDCs)are mobi
12、lized by primary tumor-derived soluble factors to thedistinct organ,leading to the formation of a pre-metastaticniche as fertilized soil in preparation for the settlement andfurther colonization of the metastatic cells.This process in-volves crosstalk among various tumor-secreted factors,thehost str
13、omal microenvironment,and mobilized and recruitedBMDCs within future metastatic sites(Erler et al.,2009;Pei-nado et al.,2012;Psaila and Lyden,2009).The mechanismsfor bilateral interaction between the primary tumor and distinctorgans to prime a favorable local microenvironment as a pre-metastatic nic
14、he is an important issue that needs to be fullyunderstood.Different types of BMDCs have been identified as contributingto the stromal remodeling in the pre-metastatic niche(Hiratsukaet al.,2006;Kaplan et al.,2005).However,how host stromalcells in distinct organs,such as epithelial cells in the lung,
15、cansense tumor-derived signals to trigger such BMDC recruitmentSignificanceFormation of the pre-metastatic niche is critical for organ-specific metastatic tropism.Using spontaneous metastaticmodels,we uncovered a significant role of host lung epithelial cell TLR3 in promoting lung pre-metastatic nic
16、he formationvia tumor exosome-mediated neutrophil recruitment.Small nuclear RNAs enriched in tumor exosomes activated TLR3 inalveolar type II epithelial cells,consequently inducing chemokine secretion and promoting neutrophil recruitment in thelung.In human lung cancers,high expression of TLR3 in ad
17、jacent tissues was closely correlated with a high level of neutro-phil infiltration,both of which predicted poor survival of the patients.Thus,our findings provide mechanistic insight for pre-metastatic niche formation and organ-site-specific tropisms of metastasis.Cancer Cell 30,243256,August 8,201
18、6 2016 Elsevier Inc.243for the pre-metastatic niche formation remains poorly defined.The epithelial surface of the lungs,lined by type I and type IIepithelial cells,serves as a biological barrier in the respiratorytract(Wagner and Griffith,2010).In addition to gas exchangeand surface tension mainten
19、ance,lung epithelial cells alsoplay an essential role in the recognition of pathogen-andinjury-associated signals,orchestrating innate immunity inlung to maintain pulmonary homeostasis(Whitsett and Alen-ghat,2015).Activation of various pattern-recognition receptors(PRRs)in these cells and subsequent
20、 production of cytokinesand chemokines induces professional immune cell infiltration(Hartl et al.,2007;Juncadella et al.,2013).Aberrant pro-inflammatory responses to stimuli in lung epithelial cells alsocontribute to chronic inflammation,indicating a potential rolein tumorigenesis(Brody and Steiling
21、,2011;Takahashi et al.,2010).However,little is known about the regulation of local tu-mor metastasis by lung epithelial cells.PRRs are responsible for sensing the presence of molecularcomponents from invading microorganisms or endogenousdamaged cells,serving as the sensors for innate immunity.Asone
22、of the originally identified and best-characterized PRRs,Toll-like receptors(TLRs)have been shown to recognize variousinflammatory innate signals and contribute to the innate defense(Akira and Takeda,2004;Cao,2016).Although both anti-tumorand tumor-promoting roles of TLRs have been reported,TLRshave
23、 gradually been found to promote multiple tumor process,including tumorigenesis,tumor growth,and metastasis(Pradereet al.,2014).Host TLRs initiate chronic inflammation to promotetumorigenesis(Dapito et al.,2012;Scheeren et al.,2014),whiletumor cell TLRs provide intrinsic anti-apoptotic and prolifera
24、tivesignals to support tumor growth and invasion(Cherfils-Viciniet al.,2010;He et al.,2007;Wang et al.,2008).The mechanismsfor how host TLRs modulate tumor metastasis attract muchattention.For example,tumor-produced factors can activateTLR2:TLR6 complexes on myeloid cells to induce tumor necro-sis f
25、actor a secretion and thus promote tumor lung metastasis(Kim et al.,2009).Tumor-secreted microRNAs bound to murineTLR7 and human TLR8 in immune cells to trigger tumor metas-tasis(Fabbri et al.,2012).However,owing to different metastaticmodels and distinct cells used,the roles of host TLRs in cancerm
26、etastasis are complex and even controversial(Pradere et al.,2014).Therefore,we set out to determine how host stromalcell TLRs,once activated by tumor-derived factors,contributeto initiate pro-metastatic inflammatory responses and pre-meta-static niche formation.RESULTSTLR3 Deficiency Prevents Lung P
27、re-metastatic NicheFormation and MetastasisTo obtain an overall view of TLRs except for well-defined TLR2and TLR7/TLR8 in the metastatic process,we subcutaneouslyinoculated Tlr3?/?,Tlr4?/?,and Tlr9?/?mice with Lewis lung car-cinoma(LLC)or B16/F10 melanoma cells and then observed thelung metastasis i
28、n the spontaneous metastatic mouse model bysurgically removing the primary tumors(Figures S1A and S1B).We found that Tlr3?/?mice,but not Tlr4?/?and Tlr9?/?mice,showed significant reduction in lung metastasis compared withwild-type(WT)littermates(Figures 1A,1B,and S1CS1E).Furthermore,Tlr3?/?mice surv
29、ived much longer than WT litter-mates after tumor removal(Figure 1C).However,no differencewas observed in early primary tumor growth in these mousemodels(Figure 1D).This observation indicated that deficiencyof TLR3 impairs cancer lung metastasis.The pre-metastatic niche plays a critical role in the
30、metastaticprocess as it prepares the congenial soil in distinct organs fortumor metastasis(Kaplan et al.,2005;Sceneay et al.,2013).Toexamine whether TLR3 could promote pre-metastatic niche for-mation in the lung,we assessed lung expression of the nichecharacteristic genes,including Bv8(also known as
31、 Prok2),S100a8,S100a9,and Mmp9,which are reported to promote tu-mor cell invasion,migration,and colonization in the metastaticsite(Wu et al.,2015).We found that the lung from Tlr3?/?miceshowed significantly lower expression of Bv8,S100a8,S100a9,and Mmp9 after tumor inoculation(Figure 1E).Furthermore
32、,fibronectin,a protein involved in the adhesion of BMDCs andtumor cells in the pre-metastatic niche(Erler et al.,2009),wasdownregulated in Tlr3?/?mice(Figure 1F).Thus,TLR3 maycontribute to lung pre-metastatic niche formation.TLR3 Deficiency Reduces Neutrophil Recruitment to theLungBMDCs are recruite
33、d to metastatic organ sites to form a meta-static niche(Kaplan et al.,2005).Thus,we analyzed the cellularcomposition of the lung in Tlr3?/?and WT littermates after tumorinoculation.In WT mice,fluorescence-activated cell sorting(FACS)analysisshowedthatCD11b+myeloidcellswereremark-ably expanded in the
34、 pre-metastatic lung,among which CD45+CD11b+Ly6G+Ly6Cintneutrophils were the most dominant(Fig-ures 2A,2B,and S2A).Ly6G?Ly6C+monocytes and F4/80+macrophages showed a moderate accumulation in lung(FiguresS2C and S2D),while CD11c+Ia/e+dendritic cells showed no dif-ference(Figure S2E).However,both the
35、percentage and numberof neutrophils in pre-metastatic lung,as well as peripheral bloodand spleen,were significantly reduced in Tlr3?/?mice comparedwith WT littermates(Figures 2A,2B,and S2B).These data indi-cate that neutrophils may be the mostprominent in pre-metasta-tic niche formation,although oth
36、er myeloid cells may be alsoinvolved.Indeed,there was still an increase of neutrophils inthe Tlr3?/?mice upon tumor inoculation compared with thatwithout tumor inoculation(Figure 2A),suggesting that otherTLRs,although less profound than TLR3,may also participatein the recruitment of neutrophils.It h
37、as been shown that neutrophils may facilitate cancer lungmetastasis(Bald et al.,2014).cKIT+and VEGFR1+myeloid cellsare the main BMDCs identified in the metastatic niche(Coffeltet al.,2015;Kaplan et al.,2005).We found that VEGFR1 was ex-pressed in neutrophils and monocytes,while cKIT+neutrophilswere
38、significantly expanded in tumor-bearing mice(Figure S2F).BV8 is highly expressed in pro-metastatic neutrophils,which isone of the major components in the niche(Kowanetz et al.,2010).We detected much higher expression of Bv8 in neutro-phils(Ly6G+Ly6C+)than in Ly6G?Ly6C+or Ly6G?Ly6C?cellsin tumor-bear
39、ing mice(Figure S2G).Therefore,reduced neutro-phil recruitment and accumulation in lung may account fordecreased lung metastasis in Tlr3?/?mice.Together,thesedata suggest that TLR3 may promote pre-metastatic niche for-mation by recruiting neutrophils to the lung.244Cancer Cell 30,243256,August 8,201
40、6Activation of Lung Epithelial TLR3 Induces ChemokineProduction and Neutrophil RecruitmentTo dissect the underlying mechanisms on impaired neutrophilrecruitment in tumor-bearing Tlr3?/?mice,we first ruled outthe possibility of a defect in neutrophils themselves in Tlr3?/?mice.Neutrophils in the bone
41、 marrow showed no significant dif-ferences in the mice with or without tumor inoculation(Fig-ure S2B).It is known that chemokine and its receptor contributeto neutrophil mobilization and recruitment.However,the expres-sion of chemokine receptors(CXCR1,CXCR2,CXCR4,andCCR2)on neutrophils was much high
42、er in Tlr3?/?mice(Figures2C and S3A).Next,we detected the levels of chemokines(CXCL1,CXCL2,CXCL5,and CXCL12)that are known to berequired for neutrophil chemotaxis in the serum and bronchoal-veolar lavage fluid(BALF),and found that chemokines fromTlr3?/?mice were much lower(Figures 2D,2E,S3B,and S3C)
43、.Bone marrow reconstitution showed that Tlr3?/?mice receivingbone marrow of WT littermates exhibited lower chemokineFigure 1.TLR3 Deficiency Prevents Lung Pre-metastatic Niche Formation(A)H&E-stained lung sections and quantification of lung metastatic foci of Tlr3?/?mice or WT littermates(n=12)after
44、 LLC and B16/F10 tumor inoculation.Scalebar,5 mm.(B)Representative images and quantitative analysis of lung metastasis of Tlr3?/?mice or WT littermates detected by ex vivo luciferase-based bioluminescenceimaging after LLC and B16/F10 tumor inoculation.(C)Survival of Tlr3?/?mice or WT littermates(n=1
45、0 each)after LLC and B16/F10 tumor inoculation(p 0.001;Kaplan-Meier test).(D)Growth curves of tumors arising from inoculation with LLC or B16/F10 tumor cells.Tumor growth was measured with a digital caliper.(E)Pro-metastatic gene expression in the lung of Tlr3?/?mice or WT littermates at 2 weeks aft
46、er LLC tumor inoculation.The data were normalized to Bv8expression of WT mice as shown in the first column.b-Actin was assayed as a control.(F)Representativeimages on fibronectin-stained lung sections and quantification of fibronectin expression of Tlr3?/?mice or WT littermates at 2 weeks after LLCt
47、umor inoculation.The arrows indicate fibronectin staining.Scale bar,100 mm.Data are mean SD of one representative experiment.Similar results were seen in three independent experiments.Unpaired Students t tests unless noted.*p 0.05,*p 0.01,*p 0.001.See also Figure S1.Cancer Cell 30,243256,August 8,20
48、16245Figure 2.TLR3 Deficiency Reduces Neutrophil Recruitment to the Lung(A and B)The proportions(A)and absolute numbers(B)of neutrophils in the lung were detected by flow cytometry in Tlr3?/?mice or WT littermates after LLCtumor inoculation.(C)Fluorescence intensity of CXCR2 and CXCR4 expression on
49、neutrophils of Tlr3?/?mice or WT littermates after LLC tumor inoculation was detected by flowcytometry.(legend continued on next page)246Cancer Cell 30,243256,August 8,2016levels,less neutrophil recruitment,and less lung metastasis(Fig-ures 2F2I and S3D).These data suggest that TLR3 present onhostst
50、romalcells,butnottheTLR3onBMDCs,iscriticalforche-mokine production and neutrophil recruitment.qPCR analysis showed that Tlr3 was highly expressed in lungepithelial cells,dendritic cells,and macrophages,but not in neu-trophils and fibroblasts in the lung tissues(Figures 3A and 3B).Immunofluorescence