外泌體在腫瘤精準(zhǔn)診斷與治療中的研究進(jìn)展

胡牧仁,陳婷,楊長青,葛步軍

(1.同濟(jì)大學(xué)附屬同濟(jì)醫(yī)院普外科,上海 200065; 2.溫州醫(yī)科大學(xué)附屬第一醫(yī)院皮膚科,浙江溫州 325000; 3.同濟(jì)大學(xué)附屬同濟(jì)醫(yī)院消化內(nèi)科,上海 200065)

外泌體在腫瘤精準(zhǔn)診斷與治療中的研究進(jìn)展

胡牧仁1,陳婷2,楊長青3,葛步軍1

(1.同濟(jì)大學(xué)附屬同濟(jì)醫(yī)院普外科,上海 200065; 2.溫州醫(yī)科大學(xué)附屬第一醫(yī)院皮膚科,浙江溫州 325000; 3.同濟(jì)大學(xué)附屬同濟(jì)醫(yī)院消化內(nèi)科,上海 200065)

外泌體是一種細(xì)胞分泌的具有雙層脂質(zhì)膜結(jié)構(gòu)的納米級小囊泡,幾乎所有類型的細(xì)胞均可分泌外泌體.外泌體攜帶核酸和蛋白質(zhì)等多種具有生物活性的內(nèi)容物,不但在細(xì)胞間信息交流和信號轉(zhuǎn)導(dǎo)的過程中起著重要作用,而且參與了人體內(nèi)各種病理生理過程.腫瘤來源的外泌體參與調(diào)控腫瘤的生長發(fā)展和侵襲轉(zhuǎn)移等過程.外泌體在腫瘤的早期診斷、預(yù)后判斷以及作為治療工具方面的探索已經(jīng)成為研究熱點(diǎn).對外泌體在腫瘤的發(fā)生發(fā)展、轉(zhuǎn)移侵襲以及在腫瘤精準(zhǔn)診斷與個(gè)體化治療方面的進(jìn)展作一綜述.

外泌體;腫瘤;精準(zhǔn)診斷;治療

腫瘤是一種在分子遺傳上具有極大異質(zhì)性的疾病,因此腫瘤學(xué)科是精準(zhǔn)醫(yī)療的重要應(yīng)用領(lǐng)域之一[1-2].新興的高通量技術(shù)如組學(xué)技術(shù)的發(fā)展,使得對轉(zhuǎn)錄組學(xué)、蛋白組學(xué)和代謝組學(xué)等層次的研究已日臻成熟,為腫瘤的精準(zhǔn)治療提供了不可或缺的技術(shù)基礎(chǔ)[3].從外科手術(shù)、影像引導(dǎo)下微創(chuàng)治療到靶向藥物治療,乃至提前預(yù)知患癌幾率并進(jìn)行早期干預(yù),腫瘤的治療正在向個(gè)體化、精準(zhǔn)化的醫(yī)療模式方向發(fā)展.腫瘤精準(zhǔn)治療模式是一種基于腫瘤患者專屬特征的個(gè)體化治療,是針對腫瘤分子機(jī)制基礎(chǔ)和臨床特點(diǎn)制定的特定診療策略.為了實(shí)現(xiàn)精確診斷,除了綜合分析生活方式、環(huán)境信息等數(shù)據(jù)外,采用的主要手段是基因和蛋白檢測.腫瘤細(xì)胞外泌體作為一種攜帶來源組織細(xì)胞成分的細(xì)胞外囊泡,其攜帶的核酸和蛋白分子可作為腫瘤檢測的重要分子標(biāo)志物以及治療的新靶點(diǎn)[4-5].以外泌體作為切入點(diǎn),從分子水平篩查腫瘤患者,以及早期診斷、預(yù)測轉(zhuǎn)移復(fù)發(fā)和監(jiān)測療效等,將極大地推動(dòng)腫瘤精準(zhǔn)醫(yī)療模式的發(fā)展.

1 外泌體的形態(tài)和形成過程

外泌體(exosome)是一種在生理或者病理情況下由細(xì)胞以胞吐的形式分泌到細(xì)胞外的胞外囊泡,直徑約為40～100 nm,由脂質(zhì)雙分子層包繞而成.外泌體最早由Johnstone等[6]在研究網(wǎng)織紅細(xì)胞成熟過程中囊泡的形成時(shí)發(fā)現(xiàn).外泌體在電鏡下呈圓形小碟狀.多種類型的細(xì)胞如骨髓細(xì)胞、淋巴細(xì)胞、內(nèi)皮細(xì)胞、腫瘤細(xì)胞等均可分泌外泌體,它廣泛存在于血液、尿液、乳液、腹水、腦脊液等各種體液中[7-8].外泌體的形成是一個(gè)有序而又復(fù)雜的動(dòng)態(tài)過程.外泌體由細(xì)胞內(nèi)的多泡內(nèi)體(multivesicular endosomes,MVEs)發(fā)展而來,從細(xì)胞質(zhì)膜的內(nèi)陷出芽形成MVEs開始,選擇性地接收裝載細(xì)胞胞漿內(nèi)的核酸、蛋白及脂質(zhì)等物質(zhì)的管腔內(nèi)囊泡(intraluminal vesicles,ILVs)進(jìn)入儲(chǔ)存泡即MVEs,最后通過MVE與細(xì)胞質(zhì)膜的融合釋放到細(xì)胞外形成大小不一的外泌體[7],其中部分MVEs與溶酶體結(jié)合后被降解.另外,外泌體的形成過程中還需要Rab蛋白家族、四跨膜蛋白超家族(tetraspanins)或者轉(zhuǎn)運(yùn)必需內(nèi)體分選復(fù)合物(endosomal sorting complexes required for transport,ESCRT)的參與[9-10].

2 外泌體的生物學(xué)功能

外泌體可以與受體細(xì)胞的胞膜發(fā)生融合進(jìn)入細(xì)胞,釋放內(nèi)部信號分子進(jìn)入受體細(xì)胞發(fā)揮生物學(xué)效應(yīng),或者外泌體表面蛋白質(zhì)或脂質(zhì)配體被靶細(xì)胞表面受體識(shí)別并結(jié)合激活,進(jìn)而參與局部和遠(yuǎn)處的細(xì)胞間的信息交流及信號分子轉(zhuǎn)導(dǎo),從而調(diào)控細(xì)胞增殖、細(xì)胞分化、炎癥免疫反應(yīng)、血管新生、腫瘤侵襲與轉(zhuǎn)移及耐藥等過程[8,11].已有研究發(fā)現(xiàn),腫瘤患者血清中的腫瘤細(xì)胞釋放的外泌體攜帶的單鏈或者雙鏈DNA與原發(fā)腫瘤組織高度一致[12-13].越來越多的研究發(fā)現(xiàn)外泌體攜帶的組織細(xì)胞來源的一系列具有生物活性的功能性分子具有促進(jìn)腫瘤的生物學(xué)效應(yīng).這些活性分子主要包括核酸、蛋白質(zhì)和脂質(zhì)等[14],其中核酸如DNA(deoxyribonucleic acid)片段、信使核糖核酸(messenger ribonucleic acid,mRNA)和microRNA(miRNA),蛋白質(zhì)如白介素、整合素等細(xì)胞因子.據(jù)報(bào)道,約有19 000種mRNA,2 838種miRNA,27 000種蛋白質(zhì)和1 100種脂質(zhì)分子存在于各種類型的外泌體中[15].另外,外泌體的包裹作用使得核酸分子等能夠穩(wěn)定存在于細(xì)胞外液中,因此基于外泌體的液態(tài)活檢將為腫瘤精準(zhǔn)醫(yī)學(xué)開啟腫瘤個(gè)體化治療的新篇章,具有巨大的臨床應(yīng)用前景和研究價(jià)值.

3 腫瘤外泌體與癌前利基:預(yù)測腫瘤轉(zhuǎn)移的利器

腫瘤細(xì)胞通過血循環(huán)由原發(fā)部位擴(kuò)散轉(zhuǎn)移到遠(yuǎn)處組織器官是腫瘤患者死亡的主要元兇之一,這個(gè)過程最初被認(rèn)為是隨機(jī)選擇的結(jié)果.種子土壤學(xué)說認(rèn)為不同的腫瘤細(xì)胞(種子)會(huì)尋找特定的有利其生存的環(huán)境(土壤),因此腫瘤具有轉(zhuǎn)移到特定器官的傾向.這種特定有利的微環(huán)境稱為“pre-metastatic niche”(癌前利基).已有研究發(fā)現(xiàn),在種子到達(dá)目的器官之前,能通過分泌外泌體影響“土壤”形成癌前利基,從而幫助促進(jìn)腫瘤轉(zhuǎn)移[16].原發(fā)性腫瘤組織分泌的外泌體通過血液循環(huán)到達(dá)靶器官,幫助形成有利于晚期轉(zhuǎn)移的轉(zhuǎn)移前微環(huán)境.

轉(zhuǎn)移性黑色素瘤癌細(xì)胞通過分泌富含受體酪氨酸激酶(C-MET)的外泌體“改造”骨髓(bone marrow,BM)干細(xì)胞從而增強(qiáng)原發(fā)性腫瘤的轉(zhuǎn)移行為,以及誘導(dǎo)靶器官組織的血管滲漏和重編程BM干細(xì)胞形成有利于腫瘤細(xì)胞種植生存的環(huán)境[17].另外,已有研究發(fā)現(xiàn),腫瘤外泌體表達(dá)不同亞型的整合素參與腫瘤的器官特異性轉(zhuǎn)移,這些整合素被受體細(xì)胞接受后會(huì)增強(qiáng)其黏附能力,上調(diào)促炎基因S100的表達(dá)水平和Src磷酸化水平,從而促進(jìn)腫瘤的遠(yuǎn)處定向轉(zhuǎn)移,其中整合素α6β1和α6β4介導(dǎo)腫瘤的肺轉(zhuǎn)移,αvβ5介導(dǎo)腫瘤的肝轉(zhuǎn)移[18].骨向性腫瘤細(xì)胞在攝取嗜肺腫瘤細(xì)胞來源的外泌體后能重新定向轉(zhuǎn)移到肺部組織.除了外泌體整合素,腫瘤細(xì)胞外泌體攜帶的miRNA和Tetraspanins也參與改造轉(zhuǎn)移前宿主器官基質(zhì)細(xì)胞[18-19].此外,轉(zhuǎn)移宿主位點(diǎn)的細(xì)胞除了攝取原發(fā)腫瘤來源外泌體影響腫瘤的定向轉(zhuǎn)移外,其自身還能分泌外泌體以幫助“種子”腫瘤細(xì)胞的定植生存.黑色素瘤細(xì)胞抑癌基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)在經(jīng)血運(yùn)轉(zhuǎn)移到腦組織時(shí)失去正常表達(dá).主要是腦星形膠質(zhì)細(xì)胞分泌的外泌體通過攜帶miR-19a可逆性調(diào)控腫瘤細(xì)胞內(nèi)PTEN的表達(dá),進(jìn)而影響chemokine ligand 2(CCL2)和nuclear factor κB(NF-κB)相關(guān)通路,增強(qiáng)腫瘤細(xì)胞的增殖和抵抗凋亡的能力,為腫瘤細(xì)胞的轉(zhuǎn)移定植提供環(huán)境基礎(chǔ)[20].由以上結(jié)果可知,腫瘤細(xì)胞來源的外泌體可以作為預(yù)測轉(zhuǎn)移靶器官的生物標(biāo)志物.

4 外泌體與腫瘤診斷

腫瘤的早期診斷與及時(shí)治療是腫瘤患者提高預(yù)后的關(guān)鍵.然而,目前臨床上尚缺乏有效的早期診斷方法,亟需尋找新型策略以發(fā)現(xiàn)可以應(yīng)用于腫瘤早期診斷的生物標(biāo)志物[21-22].腫瘤外泌體作為溝通腫瘤細(xì)胞間通訊信息交換的生物載體,所攜帶的一些核酸分子和特異性蛋白等為腫瘤的早期診斷和治療提供了新的靶標(biāo).通過基因組、蛋白質(zhì)組等組學(xué)技術(shù)獲得患者體內(nèi)生物學(xué)信息并結(jié)合臨床特點(diǎn),可以在分子水平診斷疾病,從而實(shí)現(xiàn)新型精準(zhǔn)醫(yī)療腫瘤個(gè)體化治療[1].

4.1 外泌體核酸:腫瘤標(biāo)志物

通過鑒定分析發(fā)現(xiàn),miRNA和mRNA是外泌體核酸的主要成分[23-24],已有研究也表明外泌體含有其他RNA,如轉(zhuǎn)運(yùn)RNA(transfer RNA,tRNA)和長鏈非編碼RNA(long noncoding RNA,lncRNA)等[25],以及單鏈或雙鏈DNA片段[13,26].外泌體中的核酸分子是最受關(guān)注的一類可作為腫瘤診斷生物標(biāo)志物的分子,這是由于外泌體的包裹作用使得外泌體不易被RNA酶降解從而能穩(wěn)定存在于細(xì)胞外液中[27-29].在胰膽管癌患者的唾液樣本中提取的外泌體中,miR-4644和miR-1264的表達(dá)水平顯著高于正常健康人[30].此外,黑色素瘤抗原基因1(melanoma antigen-1,MAGE-1)以及人類表皮生長因子受體2(human epidermal growth factor receptor-2,HER2)/neu的mRNA水平在胃癌患者血漿中提取的外泌體中顯著升高[31].Taylor等[27]發(fā)現(xiàn)miR-21,miR-141,miR-200a,miR-200b,miR-200c,miR-203,miR-205和miR-214這8種miRNA可作為卵巢癌診斷標(biāo)志物,同時(shí)8種miRNA也存在于源自卵巢癌患者的循環(huán)腫瘤外泌體中.上述結(jié)果表明,外泌體尤其是存在于循環(huán)外泌體中的核酸分子具有作為腫瘤早期診斷標(biāo)志物的潛力.研究人員對腫瘤活檢樣品進(jìn)行miRNA分析,發(fā)現(xiàn)在肺癌患者中miRNA與正常對照組顯著不同,然而與腫瘤組織活檢樣本之間具有高度的相似性[32],這說明循環(huán)外泌體miRNA可為肺腺癌的液體活檢提供一種新的思路和方向.另外,外泌體中的miRNA水平與腫瘤生長進(jìn)展和侵襲性相關(guān),惡性食管鱗狀細(xì)胞癌(esophagealsquamous-cell carcinoma,ESCC)患者中外泌體miR-21顯著高于良性患者[33],這表明外泌體不僅可以作為生物標(biāo)志物,也可以作為一個(gè)新的治療靶點(diǎn).近年來,隨著外泌體提取技術(shù)的成熟和應(yīng)用,基于RNA測序的miRNA分析已成功地在多種腫瘤模型包括胰腺癌、胃癌、乳腺癌、結(jié)腸癌、前列腺癌和膠質(zhì)母細(xì)胞瘤中發(fā)現(xiàn)了可用作候選診斷標(biāo)志物的多種外泌體(見表1).

表1 外泌體核酸作為腫瘤診斷的生物標(biāo)志物Table 1 Exosomal nucleic acids as biomarkers for cancer diagnosis

4.2 外泌體蛋白:腫瘤標(biāo)志物

由于外泌體特殊的結(jié)構(gòu)和溝通細(xì)胞間通訊的功能,外泌體表面蛋白同樣可作為腫瘤生物標(biāo)志物.外泌體含有能反映其來源細(xì)胞的多種蛋白質(zhì)分子.在乳腺癌患者血清中的外泌體中,分化抗體群(cluster of differentiation 24,CD24)的表達(dá)水平顯著上調(diào)[37].接受手術(shù)的乳腺癌患者外泌體中的EDIL3(EGF-like repeats and discoidin I-like domains 3)和纖連蛋白的表達(dá)水平顯著降低[38-39],這說明循環(huán)外泌體中的EDIL3和纖連蛋白可以作為反映治療反應(yīng)性的標(biāo)志物,即可以用于動(dòng)態(tài)評估患者預(yù)后.細(xì)胞表面蛋白多糖Glypican-1存在于早期和晚期胰腺癌患者的血清外泌體中,但并不存在于良性胰腺疾病患者的血清外泌體中[40].對良性腫瘤患者和惡性卵巢癌患者血漿中分離的外泌體進(jìn)行差異分析,發(fā)現(xiàn)后者攜帶MAGE 3/6和TGF-β1(transforming growth factor beta 1)[41],以上結(jié)果表明可以通過檢測腫瘤外泌體裝載的蛋白分子來進(jìn)行早期診斷,并已在多種腫瘤類型外泌體中發(fā)現(xiàn)并驗(yàn)證(見表2).

表2 外泌體蛋白作為腫瘤診斷的生物標(biāo)志物Table 2 Exosomal proteins as biomarkers for cancer diagnosis

5 外泌體與腫瘤治療

外泌體作為具有脂質(zhì)膜結(jié)構(gòu)的天然納米級囊泡,能夠攜帶內(nèi)源性或外源性核酸分子和蛋白質(zhì)分子等,被靶細(xì)胞攝取后調(diào)控影響腫瘤細(xì)胞的增殖、轉(zhuǎn)移侵襲等生物學(xué)行為.此外,外泌體還可以被改造成攜帶有效治療物質(zhì)如蛋白質(zhì)或RNA藥物的高效運(yùn)載體,利用人體細(xì)胞自身精準(zhǔn)通訊系統(tǒng)將其傳遞到機(jī)體特定部分,從而實(shí)現(xiàn)腫瘤的基因靶向治療[45-47].因此,探索將外泌體用作治療工具有望成為腫瘤治療的一個(gè)重要方向.

在Ⅰ期臨床試驗(yàn)中,轉(zhuǎn)移性黑色素瘤患者皮內(nèi)和皮下注射源自樹突狀細(xì)胞(dendritic cells,DCs)的外泌體并加載MAGE腫瘤抗原肽治療4周,雖然尚未觀察到顯著的療效,但卻證實(shí)了外泌體治療的安全性和可行性[48].在另外一個(gè)第一階段的Ⅰ期臨床試驗(yàn)中,非小細(xì)胞肺癌患者在接種攜帶腫瘤抗原肽的自體DCs外泌體后抗腫瘤免疫反應(yīng)激活,使得疾病進(jìn)展減緩[49].已有研究表明,外泌體interferon-γ(IFN-γ)能夠增強(qiáng)免疫反應(yīng)和抑制腫瘤的生長發(fā)展[50].基于此Ⅰ期和臨床前結(jié)果,Ⅱ期臨床試驗(yàn)顯示晚期非小細(xì)胞肺癌患者在注射DCs源性IFN-γ外泌體后能夠增強(qiáng)自然殺傷細(xì)胞(natural killer cell,NK)介導(dǎo)的抗腫瘤免疫反應(yīng)的免疫[51].還有研究表明,當(dāng)外泌體裝載阿霉素的DCs來源的外泌體后,能通過整合素相關(guān)途徑到達(dá)靶細(xì)胞(如黑色素瘤細(xì)胞、乳腺癌細(xì)胞和肝癌細(xì)胞)后傳遞阿霉素到腫瘤細(xì)胞內(nèi)發(fā)揮治療作用[52].可見外泌體的裝載作用能夠極大地提高藥物的穩(wěn)定性和靶向性,從而增強(qiáng)對腫瘤的殺傷作用.

6 展望

腫瘤的晚期轉(zhuǎn)移而非原發(fā)性腫瘤常常是腫瘤患者死亡的主要原因,因此改善腫瘤患者預(yù)后和提高生存率的關(guān)鍵在于早期診斷.外泌體穩(wěn)定廣泛地存在于人體的各個(gè)組織、器官以及幾乎所有的體液中,各種類型的細(xì)胞均可釋放這些納米大小的囊泡,并且含有來源細(xì)胞的跨膜蛋白和一些胞內(nèi)蛋白如整合素或遺傳物質(zhì),它們與來源組織細(xì)胞具有極高的一致性,可見研究外泌體作為腫瘤的早期診斷和預(yù)后的生物標(biāo)志物具有重要意義.外泌體的表面蛋白能被受體細(xì)胞靶向捕獲,而外泌體的內(nèi)容物則可以改變受體細(xì)胞的生理狀態(tài).腫瘤外泌體也能通過與腫瘤微環(huán)境的其他細(xì)胞相互作用來調(diào)節(jié)腫瘤進(jìn)展、血管生成、轉(zhuǎn)移和免疫逃逸.這些由外泌體介導(dǎo)的病理過程,同時(shí)也賦予了外泌體作為生物標(biāo)志物的巨大潛能.深入了解外泌體的作用機(jī)制對于腫瘤的早期篩查、精準(zhǔn)診斷和預(yù)后評估都有著巨大價(jià)值.

除了作為生物標(biāo)志物的巨大潛能外,外泌體還為腫瘤的精準(zhǔn)治療提供了新的研究方向.為了提高抗腫瘤藥物治療的有效性,藥物裝載系統(tǒng)是一個(gè)關(guān)鍵的問題.外泌體作為一種天然的治療載體,除了裝載外源性藥物維持藥物在體內(nèi)的穩(wěn)定性外,同時(shí)還含有自身的生物活性分子,避免了免疫排斥反應(yīng).外泌體還可以利用人體細(xì)胞自身精準(zhǔn)通信系統(tǒng)將其定向移動(dòng)傳遞到目標(biāo)組織發(fā)揮治療效應(yīng),這些優(yōu)點(diǎn)可使得外泌體成為一種打破傳統(tǒng)給藥模式的理想裝載系統(tǒng),將會(huì)成為促進(jìn)腫瘤精準(zhǔn)醫(yī)療發(fā)展的一個(gè)重要工具.

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Exosomes in the precision diagnosis and treatment of cancer

HU Muren1,CHEN Ting2,YANG Changqing3,GE Bujun1
(1.Department of General Surgery,Tongji Hospital Affiliated to Tongji University, Shanghai 200065,China; 2.Department of Dermatology,First Affiliated Hospital,Wenzhou Medical University, Wenzhou 325000,Zhejiang,China; 3.Department of Gastroenterology,Tongji Hospital Affiliated to Tongji University, Shanghai 200065,China)

Exosomes are nanoscale vesicle with a double lipid membrane structure,and can be secreted by almost all types of cells.Exosomes can carry nucleic acids and proteins, and play important roles in a variety of physiological and pathological processes especially in cell communication and signal transduction.Tumor cell-derived exosomes have been proved to be involved in the regulation of tumor growth,invasion and metastasis process. Exosomes’roles in the early diagnosis,prognosis prediction and therapeutics have been a hot topic.This review summarizes the role of exosomes in the development and metastasis of cancer,and its potential function in the individually precise treatment.

exosome;tumor;precision diagnosis;treatment

R 45

A

1007-2861(2017)02-0161-08

10.3969/j.issn.1007-2861.2017.02.003

2017-04-03

國家自然科學(xué)基金資助項(xiàng)目(81370559,81370586,81670571);上海市重要疾病聯(lián)合攻關(guān)重大項(xiàng)目(2014ZYJB0201);上海市新興前沿技術(shù)聯(lián)合攻關(guān)項(xiàng)目(SHDC12014122)

葛步軍(1967—),男,主任醫(yī)師,博士生導(dǎo)師,博士,研究方向?yàn)橄滥[瘤.E-mail:gebujun@126.com