腫瘤轉移新進展哈醫大一院病理科王立峰腫瘤的侵襲和轉移概念發展概況基本過程腫瘤轉移研究的新進展癌細胞侵襲：癌細胞離開其原瘤灶組織侵犯了鄰近組織，并在該處繼續繁殖生長，這個過程稱之為侵襲。

概念：1889年，StephenPaget“種子土壤假說”：腫瘤細胞（“種子”）對靶器官（“土壤”）特異親和，后者可以為瘤細胞提供適宜微環境；通過將黑色素瘤細胞注射到小鼠后發生選擇性轉移得到證實。發展概況：1929年Ewing腫瘤轉移的“機械和解剖”學說：以器官和解剖、血流分布來解釋轉移的發生。1952年Luclee等注射鱗癌細胞到兔“特殊親和”現象已為人們所公認。相同情況下，轉移灶數目：肝內>肺內股靜脈肝靜脈內1977年Fidler在Science上發表文章提出“在母瘤中存在不同轉移力的細胞克隆”提出了腫瘤的異質性概念。腫瘤侵襲的過程腫瘤轉移的過程轉移的器官選擇性基本過程

一.腫瘤細胞的增殖和擴展：

瘤細胞的不斷增殖是腫瘤侵襲的前提

某些生長活躍的瘤細胞增殖腫瘤組織內部壓力增高

（擴張性壓力）癌細胞向壓力低的方向侵襲（有人認為惡性腫瘤組織內壓力比正常組織高，導致其侵襲能力也較強）

以進入血管、淋巴管等形式遠處轉移侵襲與增殖活性以外的其他因素

*生長較快的乳腺纖維瘤不發生侵襲*有高度侵襲能力的乳腺硬癌其生長是緩慢的

*體外實驗：

一起相互接觸進行培養（沒有產生壓力），小塊癌組織小塊癌組織癌組織侵襲正常組織

侵襲和轉移的實現還取決于：腫瘤細胞對正常組織的破壞能力腫瘤細胞的運動能力對侵襲轉移中所遭遇環境的適應性等因素。增殖活性是腫瘤侵襲、轉移的基礎和前提2.腫瘤細胞表面負電荷增多：唾液酸的羥基造成腫瘤細胞間的相斥力增加，腫瘤細胞粘附性下降

易從主瘤體上脫落3.腫瘤細胞粘附分子的表達下降：調控細胞與細胞之間的粘附作用的分子鈣粘連素。特點：它們是鈣依賴性跨膜粘附因子具有同種分子親和性促使腫瘤細胞運動的相關因子：

自分泌運動因子（AMF）、表皮生長因子（EGF）類胰島素生長因子、肝細胞生長因子、IL-1、IL-3、和IL-6等多種細胞因子，轉化生長因子（TGF）、干擾素2.溶解酶釋放：*腫瘤細胞本身產生多種分解酶溶解細胞外基質

*腫瘤細胞的一些代謝產物溶解小血管基底膜增加腫瘤細胞進入循環系統并形成轉移機會

腫瘤細胞更易進入新形成的血管（腫瘤內新生毛細血管基底膜不完整）返回四.血管生成腫瘤轉移過程腫瘤細胞侵入血管和淋巴管是其脫離原發瘤到轉移的開始階段瘤細胞在脈管中的錨定粘附溢出循環系統轉移灶形成一.瘤細胞在脈管中的錨定粘附

微小腫瘤栓子

腫瘤細胞結合到內皮層可使其收縮基底膜暴露毛細血管內皮細胞周期性頻繁脫落更新二.瘤細胞逸出循環系統腫瘤細胞逸出的屏障脈管系統內皮細胞下細胞外基質各種蛋白質碳水化合物分子

構成的致密網

與基底膜接觸后

腫瘤細胞特殊膜受體結合基質蛋白（纖維連接素、層粘連素、血小板反應素）瘤細胞與基底膜粘附分泌釋放多種蛋白水解酶，降解局部基質

瘤細胞的偽足伸入被降解的區域內，然后整個細胞隨之進入三.轉移灶形成瘤細胞穿出血管壁后能否形成轉移灶，取決于：1.瘤細胞與靶器官實質細胞的粘附作用與器官特異性有關2.瘤細胞本身增殖能力強弱

返回不同來源的瘤細胞有相同的特定多發轉移部位。相同性質但生長在不同部位的瘤細胞，轉移部位可不同。腫瘤轉移的器官選擇性瘤細胞轉移的器官特異性產生機制：1.轉移的瘤細胞與內皮細胞的特殊粘附（1）腫瘤細胞可以識別內皮細胞在表面結構、生化和功能上的差異（2）與靶器官內皮下基質的特異性粘附

2.靶器官內生長因子對腫瘤細胞生長的影響器官微環境對腫瘤細胞的轉移過程具有特定影響的實驗：給裸小鼠皮下移植人結腸癌腫瘤細胞后

部分肝切除，使肝再生腎部分切除

明顯刺激皮下移植的人結腸癌細胞生長對皮下移植的人結腸癌細胞的生長無刺激作用不同腫瘤細胞實現器官特異性轉移有賴于：（1）宿主器官本身一系列特殊性質（2）瘤細胞特殊地對宿主環境的反應（3）各種腫瘤和宿主微環境之間關系

返回進入21世紀的主要發現及研究進展腫瘤間質及宿主作用在侵襲轉移中的作用腫瘤表型轉換-EMT的作用腫瘤轉移的“干細胞”假說器官特異性轉移的新假說“pre-metastaticniche”腫瘤細胞“休眠”與蘇醒的調節機制腫瘤轉移相關基因研究(基因表達譜,全基因組分析)-轉移并非單一基因控制.腫瘤轉移可以發生在腫瘤演進的早期-利用原發瘤的早期診斷返回解構腫瘤轉移-腫瘤轉移新進展1各種不同的基因共同決定腫瘤的轉移行為。轉移具有不同的器官選擇性，“癌癥是一種自身種植性(self-seeding)疾病”1.腫瘤細胞與微環境2.干細胞，干細胞niches與腫瘤轉移3.腫瘤器官特異性轉移4.調節性RNA和翻譯調控腫瘤細胞與微環境腫瘤周邊細胞群體，即腫瘤微環境，對于腫瘤的生長與轉移有非常大的影響，傳統針對腫瘤實體的研究已不能解釋繁雜的腫瘤生物學現象，越來越多的研究者將方向轉向了腫瘤微環境對腫瘤細胞的影響.腫瘤細胞與微環境選擇200例乳腺癌原發灶轉移灶臨床標本.異體移植移植瘤模型法國居里研究所的Poupon長期從事腫瘤臨床研究,建立移植模型免疫缺陷小鼠成功建立的22例移植瘤的生物學特性，發現移植瘤與原發瘤的生物標志物檢測結果一致，基因表達譜也基本類似;其中部分移植瘤動物發生了轉移，然而移植瘤的轉移潛能與患者的轉移狀態并無相關性。對其中有代表性的移植鼠進行臨床治療研究發現移植動物與臨床患者的預后有關。RoswellPark癌癥研究所的Akakura：“SSeCKS／Gravin／AKAPl2通過腫瘤微環境特異通路抑制腫瘤轉移”SrcSSeCKS抑制腫瘤轉移降低血管生成下調VEGF的表達前列腺增生AKT激酶活性增加自發轉移傾向增加抑制（蛋白激酶C的底物）正常表達缺失提示SSeCKS通過影響腫瘤和微環境特異通路而抑制轉移的發生

Anderson癌癥中心的Boyd：采用基因誘捕技術新發現氯離子通道4(CICN4)能誘導腫瘤轉移的發生，雖然一定程度上是因為CICN4能提高細胞的移動性，但關鍵還在于其賦予細胞在酸性微環境下的生存能力，從而導致腫瘤發生侵襲和轉移。人間質細胞中某亞群TGF-B信號改變促進了前列腺的癌變Carcinoma-associatedfibroblasts(CAF)playacriticalroleinmalignantprogression.LossofTGF-βreceptorII(TGFβR2)intheprostatestromaiscorrelatedwithprostatictumorigenesis.TodeterminethemechanismsbywhichstromalheterogeneitybecauseoflossofTGFβR2mightcontributetocancerprogression,weattenuatedtransforminggrowthfactorbeta(TGF-β)signalinginasubpopulationofimmortalizedhumanprostatefibroblastsinamodeloftumorprogression.IntroductionInatissuerecombinationmodel,lossofTGFβR2functionin50%ofthestromalcellpopulationresultedinmalignanttransformationofthenontumorigenichumanprostateepithelialcelllineBPH1.MixingfibroblastsexpressingtheemptyvectoranddominantnegativeTGFβR2increasedtheexpressionofmarkersofmyofibroblastdifferentiation[coexpressionofvimentinandalphasmoothmuscleactin(αSMA)]throughelevationofTGF-β1andactivationoftheAktpathway.Incombination,thesetwopopulationsofstromalcellsrecapitulatedthetumorinductiveactivityofCAFs.TGFβR2activityinmixedstromalcellpopulationsculturedinvitrocausedsecretionoffactorsthatareknowntopromotetumorprogression,includingTGF-β1,SDF1/CXCL12,andmembersofthefibroblastgrowthfactor(FGF)andbonemorphogeneticprotein(BMP)families.Thepresentstudywasdesignedtotestwhetherstromalheterogeneityhasdirectconsequencesontumorprogressioninahumanprostatictissuerecombinationmodel.Here,wereportthatheterogeneousexpressionofTGFβR2inprostatestromalsubpopulationselicitschangesconsistentwiththemalignantphenotypeinducedbyCAF.Invivo,tissuerecombinationoffibroblastsoverexpressingTGF-β1andSDF1/CXCL12notonlyinducedtransformationofBPH1cells,butalsopromotedarobustgrowthofhighlyinvasivecells,similartoeffectsproducedbyCAFs.Whiletheprecisenatureand/ororiginoftheparticularstromalcellpopulationsinvivoremainunknown,thesefindingsstronglylinkheterogeneityinTGF-βsignalingtotumorpromotionbytumorstromalcells.ResultsCancer-associatedfibroblastscounteracttheorganizationaleffectofembryonicmesenchymalcellsonprostaticremodelingHumanprostatecancerstromalcellsshowheterogeneousTGF-βsignalingLossofTGF-βresponsivenessinasubpopulationofprostatestromalcellsaffectsepithelialproliferationandinducestumorigenicityAbsenceofTGF-βsignalinginaproportionofstromalfibroblastsinduceschangestophenotypeandintracellularsignalingOverexpressionofTGF-β1andSDF1αinbenignhumanprostatefibroblastsinducesinvivomalignanttransformationofBPH1cellsWeexaminedwhetherrUGM(raturogenitalsinusmesenchyme)couldsuppresstheepithelialmalignantphenotyperesultingfromexposuretoCAF.Totestthis,werecombinedCAFandrUGMcellsinfivedifferentratioswithBPH1epithelialcells.

A,i–v,H&EshowsdifferentiationofBPH1cellstobenigncordswhencombinedwith100%UGM.AstheratioofCAFcellsincreased,malignanttransformationofBPH1cellswasobservedandtheepitheliumbecameprogressivelylessorganizedandmoreinvasive(ii–v).ImmunofluorescencelabelingofαSMA/Vimentin,(vi–x),

γActin/SV40(xi–xv),anddesmin/cytokeratin(xvi–xx),showedamoremuscularstromasurroundingareaswithhighpercentageofrUGM(75%).MarkersofnormalstromaldifferentiationweredramaticallydecreasedwhentheratioofCAF/rUGMincreasedtomorethan50%.Hoescht33258stainingofthenucleus(blue)confirmedthehumannatureofthestromalcells.

B,grosspictureofBPH1cellsrecombinedwithrUGMorCAFcells(iandii)showingepithelialinvasionintothekidneyparenchyma.rUGMinducedbenignglandulardifferentiation,whichwasseparatedbyathicklayerofdifferentiatedSMA(＋)fibroblasts(green)fromtheunderlyingkidney.C,Tumorvolumeswerequantitated,graphicalrepresentationofthemean±SDofthegraftsisshownn=6.ThevolumeofthetumorsincreasedwiththepercentageofCAFTheseresultsshowthattheepithelialresponseisdictatedbytheoverallparacrinesignalingenvironment,anddemonstratethatthisresponsecanbemodifiedbymixingdifferentpopulationsofstromalcells.A,Immunohistochemicalstainingoftissuerecombinantsforphospho-Smad2.BPH1+CAFrecombinantsshowedthehighestproportionofphospho-Smad2positivestromalcells(50%)comparedwitheitherBPH1+BHPrS1(<10%)orBPH1+rUGM(<25%).B,phospho-Smad2expressioninhumanprostatecancertissuearray.Normalprostateepithelialcellsweresurroundedbycellsexpressinglowlevelsofphospho-Smad2.StromalcellsinthetumorsaswellasthoseinnormallookingareasinthePZ(peripheralzone)ofcancerpatientshadaphospho-Smad2SCOREofmorethan2.5representingmorethan50%ofpositivecells.C,additionofTGF-β1ligandtoculturedfibroblastsinducedtheexpressionαSMAandphospho-Smad2inasubpopulationofCAF,whereastheBHPrS1(Benignhumanprostatestromalcells)showedamorehomogeneouspatternofexpression.NotemorphologicchangestoCAFwithserumcomparedwithBHPrS.ThisphenomenamaybetheresultoftheabilityofCAFtoremodelcollagenmatrices.Interestingly,71%oftheTGF-βresponsiveCAFalsoexpressSMAsuggestingthatthehighlevelsofTGFβfoundinthecancerstromanotonlyactontumorepithelialcells,butcouldalsoexertaneffectinasubsetofstromalcellsthathaveintactTGF-βsignaling.

A,MTTassayofBPH1cellsexposedtoCAF,BHPrS1-EV,BHPrS1-DN,orBHPrS1-EV/BHPrS1-DNCM.EpithelialcellsproliferatedfasterinthepresenceofCAFandBHPrS1-EV/BHPrS1-DNCMascomparedwithmediumconditionedbytheotherfibroblasts.B,BPH1+BHPrS1-EV/BHPrS1-DN,BHPrS1-EV,orBHPrS1-DNtissuerecombinantsweregraftedintomaleSCIDmicefor8weeksbeforeharvestingthetissues.BPH1+BHPrS1-EV/BHPrS1-DNrecombinantsproducedthelargesttumorsasshownbythegrossappearance(arrowhead)andquantitationoftumorvolume.BHPrS1expressingadominantnegativeTGFβR2(BHPrS1-DNTβRⅡ),acontrolvector(BHPrS1-EV)C,HistologicalexaminationrevealedmalignanttransformationofBPH1onlywithmixedBHPrS1-EV/BHPrS1-DNstromalcells(i–iii).Theadeno-squamousphenotypewithinvasionintothekidneyresembledBPH1+CAFrecombinants.Thearrowindicatesinvasionintothehostkidney.ImmunofluorescencestainingofSV40confirmedtheinvasionofBPH1cellsintothekidneyparenchymaofBPH1+BHPrS1-EV/BHPrS1-DNgrafts,noinvasionwasseenintheotherrecombinants(iv–vi).ThepresenceofBHPrS1-DNincreasedtheproportionofKi67positivecellsintheBPH1+BHPrS1-EV/BHPrS1-DNandBPH1+BHPrS1-EVrecombinants(vii–ix).MalignanttransformationofBPH1cellswasaccompaniedbychangesinthestromalcompartmenttowardamyofibroblastphenotype,asnotedbytheincreasedexpressionofvimentinandSMA(x–xii).ThesedatasuggestheterogeneousTGF-βresponsivenessthestromacaninducemalignanttransformationoftheinitiatedprostateepithelialcelllineBPH1,inamannerconsistentwithpreviousobservationsofCAF.Molecularconsequencesofheterogeneousstromalcellmixes.BHPrS1-EV,BHPrS1-DN,andBHPrS1-EV/BHPrS1-DNmixtureswereculturedfor48hoursbeforeRNAandproteinisolation.

A,RT-PCRanalysisshowedincreasedSDF1expressionanddown-regulationofTGFβR3inBHPrS1-EV/BHPrS1-DNcomparedwiththeothergroups.B,PCRarrayanalysisvalidatedtheincreasedexpressionofTGF-β1inBHPrS1-EV/BHPrS1-DNandBHPrS1-DNcells(arrow)andshoweddysregulationofseveralgenesinvolvedintumorprogression.C,evaluationbydensitometricanalysisoftheproteinexpressionofdownstreamsignalingandpotentialparacrinemediatorsoftumorprogressionshowedincreasedTGF-signaling(exemplifiedbyphospho-Akt,SMA,andp27)andCD90inBHPrS1-EV/BHPrS1-DNmixtures.thesedatasuggestthatchangesinTGF-βsignalingmayregulatetheinteractionsbetweenstromalcellsthatmighthelpdeterminenotonlytheCAF-phenotype,butalsotheoverallparacrinesignalingenvironmentresponsibleforthetumorpromotingabilitiesofthecancerstroma.

A,RT-PCRanalysisshowedincreasedSDF1expressionanddown-regulationofTGFβR3inBHPrS1-EV/BHPrS1-DNcomparedwiththeothergroups.

B,BHPrS1-TGF-β1andBHPrS1-SDF1αwererecombinedwithBPH1cellsandxenograftedunderthekidneycapsuleforabout8weeks.ThevolumeofthetumorscomposedofBHPrS1-TGF-β1+BPH1andBHPrS1-SDF1+BPH1weresignificantlylargerthanthecontrols.Notetheinvasivecharacteristicofthetumors(arrows)C.Histologicalexaminationrevealedmalignanttrasnformationoftheepithelialcells(i–iii).TGFβ1-expressingfibroblastshadagreaterimpactontheproliferationofBPH1cellsasshownbyKi67stainingascomparedwithBHPrS1-SDF1αcells,(iv–vi).BHPrS1-EVfibroblastsexhibitedlightbluestromalTrichromestainingindicativeofsomecollageninthestroma.Incontrast,recombinantscomposedofBHPrS1-SDF1αstainedpredominantlyredsuggestingamoremuscularphenotype.IntenseblueTrichromestaininginthestromaofBHPrS1-TGFβ1+BPH1recombinantsrevealedtheextensivecollagendepositioninthesetumors(vii–ix).IntenseSDF1αstainingcorroboratedthesecretionofthechemokineingraftscomposedofSDF1α-expressingcomparedwithTGFβ1-expressingfibroblasts(x–xii).OverallourdatasuggestthatspecificstromallysecretedfactorsresultingfrompartiallossofTGFβR2canresultinparacrineprostatecancerpromotion.DiscussionWefoundthatoverexpressionofTGF-β1bynormalfibroblastswassufficienttoinduceneoplasticchanges.Similarobservationsweredescribedwithfibroblastsisolatedfromreductionmammoplasty

Theseobservationsindicatethatanalteredheterogeneousstromalenvironmentcanpromotehumanprostatecancerformationbychangesintherepertoireofsecretedfactors.Interestingly,abrogationofTGF-βsignalinginfibroblastsresultedintheincreaseofTGF-βligandsecretion.Thus,ahigherBHPrS1-DNTRII/BHPrS1-EVratiocausedmoreTGF-β1secretiontosupporttheconversiontoamyofibroblastphenotype.Inthisstudy,wedemonstratedthatheterogeneityofhumanprostatestromamaybeanimportantcontributortostromallyinducedcarcinogenesis.AbrogationofTGF-signalinginasubpopulationofprostatestromalcellsisconsistentwiththepreviouslydemonstratedlossofstromalTGFβR2inprostatecancer.Whilethebiologicalsituationisunlikelytobethissimple,thismodelrepresentsasteptowardmodelingstromalcomplexity.FurtherexplorationoftheintrinsicmechanismsresponsiblefortheconversiontotheCAFphenotype,andtheepithelialresponsestosuchaphenotype,arenecessaryforthedevelopingofeffectivecancertherapiesthatcantargetboththeepithelialandstromalcompartments.干細胞、干細胞niches與腫瘤轉移腫瘤干細胞CancerStemCells(CSC)

腫瘤中僅少數細胞具有在移植的實驗動物體內形成腫瘤的能力可以通過不同表面標記將腫瘤群體中腫瘤干細胞與其他細胞群分離開腫瘤干細胞形成的腫瘤仍然包含原腫瘤中的所有亞群。

干細胞niches干細胞所居留微環境的核心區域，影響著干細胞的增殖和分化，通過與干細胞之間的直接抑或間接作用影響干細胞的命運，它的異常可能使正常干細胞轉化為腫瘤干細胞，同時分化細胞進入niche后也可能成為腫瘤干細胞，進而啟動腫瘤的發生和發展，而干細胞的動員與歸巢又與腫瘤侵襲和轉移密切相關。niches異常使正常干細胞轉化為腫瘤干細胞正常影響干細胞增殖和分化.異常分化細胞進入niches后也可轉化為腫瘤干細胞正常通過直接或間接作用影響干細胞命運上皮-間質轉化（EMT）上皮細胞-間質轉化本身是一種基本的病理生理現象，參與胚胎發育、組織重建，上皮細胞表型的缺失及獲得間質特性為主要特征。近年來EMT因為其在腫瘤細胞侵襲和遠處轉移中的重要作用而成為腫瘤領域的研究熱點。Micewiththyroid-speci?cexpressionofoncogenicBRAF(Tg-Braf)developpapillarythyroidcancers(PTCs)thatarelocallyinvasiveandhavewell-de?nedfociofpoorlydifferentiatedthyroidcarcinoma(PDTC).ToinvestigatethePTC–PDTCprogression,weperformedamicroarrayanalysisusingRNAfrompairedsamplesofPDTCandPTCcollectedfromthesameanimalsbylasercapturemicrodissection.Analysisofeightpairedsamplesrevealedaprofoundderegulationofgenesinvolvedincelladhesionandintracellularjunctions,withchangesconsistentwithanepithelial–mesenchymaltransition(EMT).Thiswascon?rmedbyimmunohistochemistry,asvimentinexpressionwasincreasedandE-cadherinlostinPDTCcomparedwithadjacentPTC.Moreover,PDTCstainedpositivelyforphospho-Smad2,suggestingarolefortransforminggrowthfactor(TGF)βinmediatingthisprocess.

Accordingly,TGFβ-inducedEMTinprimaryculturesofthyroidcellsfromTg-Brafmice,whereaswild-typethyroidcellsretainedtheirepithelialfeatures.TGFβ-inducedSmad2phosphorylation,transcriptionalactivityandinductionofEMTrequiredmitogen-activatedproteinkinase(MAPK)pathwayactivationinTg-Brafthyrocytes.Hence,tumorinitiationbyoncogenicBRAFrenders

thyroidcellssusceptibletoTGFβ-inducedEMT,throughaMAPK-dependentprocess.ResultsGeneexpressionprofilesofPTCandPDTCfromTg-BrafmiceEMToccursduringprogressionofPTCtoPDTCMarkedincreaseinpSmad2inPDTCTGFβ-inducedEMTrequiresmitogen-activatedproteinkinasekinase(MEK)activationinTg-BrafthyrocytesTGFβ-inducedSmad2phosphorylationandtranscriptionalactivityisMEKdependentEMTinhumanPTCprogressiontoundifferentiatedthyroidcancersLasercapturemicrodissection(LCM)ofPDTCandPTCinTg-Brafmice.(A)(a)HematoxylinandeosinstainingofathyroidfromaTg-BrafmousereplacedbyPTC(blackarrow)andcontainingfociofPDTC(whitearrows)(100×).(b)MitoticcellinafocusofPTDC(blackarrow)(400×).GeneexpressionprofilesofPTCandPDTCfromTg-BrafmiceRepresentativeimagesofthyroidfromTg-BrafmicebeforeandafterlasercaptureofdiscreteregionsofPTCandPDTCstainedwithHistoGeneLCMFrozenSectionStainingKit(ArcturusBioscience).GeneexpressionprofilesofPTCandPDTCfromTg-BrafmiceRNAwasisolatedfromthelasercapturedcellsofPTCandPDTCpairedsamples,ampli?ed,labeledwithCy5orCy3,andco-hybridizedtothemicroarraychips.Thisidenti?ed1630geneswithsigni?cantexpressionchanges(P<0.05,falsediscoveryrate<0.1).Ofthese,955geneproductsdecreasedand675increasedinthePDTCcomparedwiththePTC.Genesinvolvedintightjunctions,desmosomes,adherentjunctions,intermediate?lamentsorbasementmembranethataredifferentiallyexpressedinmousePDTCcomparedtoPTCEMToccursduringprogressionofPTCtoPDTCTheseexpressionchangesindicatethatanEMToccurredduringprogressionfromPTCtoPDTC.(a)ArepresentativethyroidfromaTg-BrafmouseentirelyreplacedwithPTCandharboringmultiplefociofPDTC(indicatedbyarrows)stainedwithhematoxylinandeosin(i,ii),E-cadherin(iii,iv),vimentin(v,vi)orpSmad2(vii,viii)at40x(i,iii,v,vii)andthePDTCat200x(ii,iv,vi,viii).Imagesinpanels(vii,viii)wereacquiredusingtheNuanceimagingsystem,whichconvertedthehematoxylinbluecounterstaintored,andthebrownpSmadstaintoblue,toallowbetterdistinctionofpSmadfromthecounterstain.EMToccursduringprogressionofPTC

toPDTCToprovideanindependentmeasureofTGFβpathwayactivationinPDTC,weperformedimmunohistochemistryforphospho(S465/467)Smad2,andfoundamarkedincreaseinpSmad2positivecellsinPDTCcomparedwithadjacentPTC。tumor-associatedmacrophages(TAMs),whichareaknownsourceofTGFβ(Savageetal.,2008),arepresentinhighnumbersinhumanPDTCandanaplasticthyroidcancers(Ryderetal.,2008).(b)ArepresentativePTCandPDTCfromaTg-Brafmicestainedfortheactivatedmacrophagemarker,MAC-2(200X).MarkedincreaseinpSmad2inPDTC(c)Barsrepresentβ-actinnormalizedmRNAlevelsofF4-80andTginTAMsandthyroidcancercells,respectively,isolatedfromLSL-BrafV600E/TPO-Cre/ROSA26-EGFPfifthyroidsusingcellsorting.(d)Barsrepresentβ-actinnormalizedmRNAlevelsofTGFβinwild-typethyroidtissue,Tg-BrafPTCs,isolatedTAMsorisolatedBraf-expressingthyroidcancercells.MarkedincreaseinpSmad2inPDTCLSL-BrafV600E/TPO-Cremice,whichharboralatentoncogenicBrafknock-inalleleactivatedinthyroidcellsviaCre-mediatedrecombination(Francoetal.,2011),werecrossedwithROSA26-EGFPf/fmice(Maoetal.,2001)resultinginexpressionofgreen?uorescentproteininonlytheBraf-transformedthyroidcells,whichallowed?uorescence-activatedcellsortinganalysisofthyroidcancerandstromalcellsub-populations.TGFβ-inducedEMTrequiresmitogen-activatedproteinkinasekinase

(MEK)activationinTg-BrafthyrocytesIncubationwithTGFβinducedaspindle-shapemorphologyinTg-Brafthyroidcells(a),butnotwild-typethyroidcells(b),whichwasassociatedwithincreasedvimentinandlossofE-cadherinimmunostaining.U0126（MEKinhibitor）(a,b)ThyroidcellsisolatedfromTg-Braf(a)orwild-type(b)micewereplatedintochamberslidescoatedwithcollagenandincubatedfor48h.CellswerethenincubatedintheabsenceorpresenceofTGFβ(10ng/ml)withorwithoutU0126(25mM)for6days,withamediumchangeevery2days.Cellsintheleftpanelsof(a,b)wereco-stainedforE-cadherin(green)andcytokeratin(red).Cellsintherightpanelwerestainedforvimentin(green)andcytokeratin(red).Nucleiwerestainedwith4,6-diamidino-2-phenylindole(DAPI)(blue).TGFβ-inducedEMTrequiresmitogen-activatedproteinkinasekinase(MEK)activationinTg-Brafthyrocytes(c)Tg-BrafthyroidprimarycellswereplatedintoCellBindplatesandincubatedfor48h.CellswerethenincubatedintheabsenceorpresenceofTGFβfor10daysinserum-freemedium.RNAwasisolatedandusedinquantitativeRT–PCRreactionsfortheindicatedtranscripts.constitutiveMAPKactivationmayberequiredforTGFβtoinduceEMTincellstransformedbyoncogenicBraf.additionoftheMEKinhibitorU0126bluntedtheTGFβ-inducedEMT,asshownbythelackofeffectoncellmorphologyandunchangedvimentinorE-cadherinlocalizationvimentinandE-cadherinmRNAlevelswereregulatedreciprocallybyTGFβTGFβ-inducedSmad2phosphorylationandtranscriptionalactivityisMEKdependent(a)Tg-BrafthyroidcellswereplatedintoprimarycultureinCellBindplatesandincubatedfor48h.CellswerethenincubatedintheabsenceorpresenceofU0126(25mM)for3h.TGFβ(10ng/ml)wasthenaddedfor1h.Proteinlysateswerepreparedandsubjectedtowesternblottingfortheindicatedproteins.(b)ImmortalizedmBraf-p53cellswereincubatedintheabsenceorpresenceofU0126(25mM)for1–3h.TGFβ(10ng/ml)wasthenaddedfor1h.Proteinlysateswerepreparedandsubjectedtowesternblottingfortheindicatedproteins.TGFβ-inducedSmad2phosphorylationandtranscriptionalactivityisMEKdependent(c)Tg-Braforwild-typethyroidcellswereplatedintoprimarycultureinCellBindplatesandincubatedfor48h.CellswerethenincubatedintheabsenceorpresenceofTGFβ(10ng/ml)withorwithoutU0126(25iM)for24h.Proteinlysateswerepreparedandsubjectedtowesternblottingfortheindicatedproteins.TGFβ-inducedSmad2phosphorylationandtranscriptionalactivityisMEKdependent(d,e)Primarythyroidcellsfromwild-type(d)orTg-Braf(e)micewereplatedinto24-wellplatescoatedwithcollagenandincubatedfor48h.Cellswerethenco-transfectedwith3TP-luxandcytomegalovirus(CMV)-renillalucerifase.At16haftertransfection,themediumwaschangedtomediumwithorwithoutTGFβ(10ng/ml)andU0126(25mM)andincubatedfor36h.Fire?yandrenillalucerifaseactivitywerethendetermined.Barsrepresentfold-changefromuntreatedcellsin?re?ylucerifaseactivityafternormalizingfordifferencesinrenillalucerifaseactivity,andsubtractingbackgroundactivityasdeterminedincellstransfectedwithpGL3-basic.treatmentofprimaryculturesofTg-BrafthyrocyteswithTGFβresultedinincreasedlevelsofphospho(S465/467)Smad2,C-terminalsitesknowntobephosphorylatedfollowingactivationoftheTGFβreceptor,andtoberequiredforitstranscriptionalfunction(a).pretreatmentwiththeMEKinhibitorbluntedTGFβ-inducedSmadphosphoS465/467inatime-dependentmanner(b).TotalSmadlevelswerehigherincancercellsthaninwild-typethyrocytes(c).TGFβinducedfargreater3TP-Lux(TGFβ-Smadtranscriptionalreporter)

reporteractivityinTg-Braf(sevenfold)comparedwithwild-typethyroidcells(twofold)(dande).EMTinhumanPTCprogressiontoundifferentiatedthyroidcancersLossofE-cadherinstaininginhumananaplasticthyroidcancers:representativeE-cadherinstainingofahumanATC

(anaplasticthyroidcanceror(PDTC))withanadjacentregionofPTC.PTCcellsshowstrongmembraneE-cadherinstaining,whichisentirelylostinthesurroundingATCcells.OpenarrowindicatesPTCandsolidarrowindicatesanareaofATC.Left:100X,right:400X.腫瘤器官特異性轉移自從StephenPaget觀察到乳腺癌轉移到特異的組織器官，表明轉移并不是一個隨機的過程，而是腫瘤細胞(“種子”)對靶器官(“土壤”)特異親和，后者可以為瘤細胞提供適宜的微環境，這就是著名的“種子與土壤假說”，這一假說后來通過將黑色素瘤細胞注射到小鼠后發生選擇性轉移而得到證實。weused2D-DIGE(DifferenceinGelElectrophoresis)proteomicanalysisfollowedbyLC-tandemmassspectrometrytoidentifytheproteinsdifferentiallyexpressedinbrain-targetingbreastcarcinomacells(MB231-Br)comparedwithparentalMDA-MB-231cellline.Brainisacommonsiteofbreastcancermetastasisassociatedwithsignificantneurologicmorbidity,decreasedqualityoflife,andgreatlyshortenedsurvival.However,themolecularandcellularmechanismsunderpinningbraincolonizationbybreastcarcinomacellsarepoorlyunderstood.

Remarkably,highlyrelatednetworkswererevealedbytheIPAanalysisofalistof19brain-metastasis-associatedproteinsidentifiedrecentlybythegroupofDr.A.SierrausingMDA-MB-435-basedexperimentalsystem(Martinetal.,JProteomeRes20087:908–20),ora17-geneclassifierassociatedwithbreastcancerbrainrelapsereportedbythegroupofDr.J.Massaguebasedonamicroarrayanalysisofclinicallyannotatedbreasttumorsfrom368patients(Bosetal.,Nature2009459:1005–9).Betweenthetwocelllines,weidentified12proteinsconsistentlyexhibitinggreaterthan2-fold(p<0.05)differenceinexpression,whichwereassociatedbytheIngenuityPathwayAnalysis(IPA)withtwomajorsignalingnetworksinvolvingTNFα/TGFβ-,NFkB-,HSP-70-,TP53-,andIFNγ-associatedpathways.Thesefindings,showingthatdifferentexperimentalsystemsandapproaches(2D-DIGEproteomicsusedonbraintargetingcelllinesorgeneexpressionanalysisofpatientsampleswithdocumentedbrainrelapse)yieldhighlyrelatedsignalingnetworks,suggeststronglythatthesesignalingnetworkscouldbeessentialforasuccessfulcolonizationofthebrainbymetastaticbreastcarcinomacells.Results2D-DIGEanalysisofMB-231-BrandMB-231-PacelllinesIdentificationofdifferentiallyexpressedproteinsWesternblotanalysisofdifferentiallyexpressedproteinsSignalingnetworksassociatedwiththeproteinsdifferentiallyexpressedinMB-231-BrcellsComparisonwithbiologicalpathwaysassociatedwithbreastcarcinomabrainmetastasis