生物谷专访美国安德森癌症中心余棣华教授

编者按：2013年，发现细胞囊泡运输的调节机制的科学家们，荣获了当年诺贝尔生理学或医学奖。作为人体内一类重要囊泡，外泌体（exosomes）已成为科研热点。

本次生物谷和外泌体之家联合采访了美国德州大学安德森癌症中心分子肿瘤细胞学系主任，终身教授，美国科学发展学会院士和美国德州教育学院院士——余棣华教授，余教授还将出席生物谷主办的《2016外泌体与疾病研讨会》。

余棣华教授2015年10月在国际著名杂志《Nature》上发表了题为“Microenvironment-inducedPTEN loss by exosomal microRNA primes brain metastasis outgrowth”的论文，引起了学界的广泛关注。生物谷和外泌体之家就外泌体与肿瘤治疗、临床应用等相关问题对余教授进行了专访。

鸣谢：本次采访问题搜集工作由外泌体之家协助完成；汉语版全文由外泌体之家协助编译整理。

生物谷：关于您Nature文章：脑微环境中存在如此促进肿瘤定植的因子，您认为会不会是一种普遍现象，是否可能存在很多这种类似的miR。肿瘤一旦突破血脑屏障就不再受抑制？

回答：我们的研究表明，星形胶质细胞来源的miR-19a抑制转移性肿瘤细胞PTEN表达，从而促进脑转移灶的生长。乳腺癌和黑色素瘤脑转移都涉及这种机制。miR-19a是下调脑转移细胞中PTEN的microRNA之一，我们预计，星形胶质细胞来源的其他一些靶向PTEN的microRNA可能有类似miR-19a一样的功能。

星形胶质细胞来源的miRs调控转移的肿瘤细胞基因表达的机制或许同样参与了其他神经系统疾病中的PTEN丢失。一旦转移的肿瘤细胞穿过血脑屏障，脑微环境会对肿瘤定植和生长同时发挥抑制和促进作用。脑转移瘤在它复发之前可以有很长的休眠期，这意味着肿瘤成功定植和生长需要转移的肿瘤细胞和脑转移微环境之间的动态交流。一旦转移的肿瘤细胞成功地与脑微环境"谈判"并逆转脑转移微环境为利于肿瘤细胞生长条件，转移性肿瘤将过度生长而产生病症。

生物谷：您怎么看外泌体在肿瘤治疗方面的应用？

回答：外泌体被认为是一种天然脂质体的生物载体。它们具有膜渗透性，耐受性良好，并且可以穿过血脑屏障。因此，外泌体是一种很好的可用于癌症和其他疾病的治疗候选载体。最具有吸引力的外泌体应用就是利用外泌体递送药物，microRNAs，siRNAs和其它治疗化合物，因为这些成分在外泌体中更稳定^[1]。

例如，外泌体可用于递送肿瘤抑制性microRNAs或siRNAs用以敲低癌基因从而抑制肿瘤的生长。外泌体可以靶向特定的细胞类型或组织，这样的外泌体输送可能会提高疗效，减少脱靶效应。使外泌体表面携带靶向性配体可以进一步提高它们的细胞或组织靶向特异性。例如，外泌体被工程化表达Lamp2b和肿瘤靶向性整合素以增强肿瘤特异性摄取^[2]。

其次，外泌体可用于制作肿瘤疫苗。外泌体可提供肿瘤来源的抗原来诱导抗肿瘤的免疫反应。树突状细胞来源的外泌体可诱导抗肿瘤免疫并且已被应用于临床试验^[3-5]。

第三，由于肿瘤细胞来源的外泌体可修饰转移前微环境^[6]，从循环系统中去除这些外泌体可能可以阻止癌症的转移。在循环系统中减少外泌体可以利用相关抑制剂抑制肿瘤细胞外泌体的组装和释放，或通过体外净化法消除癌症患者循环中的外泌体来实现。

综上所述，尽管技术方面尚有挑战，但我们仍然可以乐观地将外泌体作为癌症治疗的新阵地。许多研究者已经在体外和体内获得证据支持外泌体用于癌症治疗的应用^[7]。然而，在癌症治疗的应用上，外泌体目前仅有少数临床试验实施。未来的研究应该明确外泌体的复杂机制以更有效地用于临床试验。

生物谷：分泌外泌体的主细胞和其他细胞都可以吞噬或者接受外泌体携载的活性分子如mRNA，miRNA等，主细胞与其他细胞对外泌体摄取或者结合哪个更有优势？可能机制是什么？

回答： 几乎所有的哺乳动物细胞在体内都可以分泌外泌体，并且也许多细胞可以摄取含有mRNA，miRNA等外泌体。肿瘤细胞，成纤维细胞，免疫细胞，星形胶质细胞和其它细胞都被报道可以释放或摄取外泌体^[7-9]。

事实上，供体细胞可再吸收它们自身分泌的外泌体。例如，在体外实验中，由胰腺肿瘤细胞系产生的外泌体具有自分泌作用可作用于自身^[10]。然而，外泌体更重要的作用似乎是通过旁分泌机制调控微环境。宿主细胞分泌的那些外泌体可作为受体细胞的外界刺激，并改变受体细胞信号传导以及宿主细胞周围的微环境。可以想到宿主细胞本身或受体细胞获取外泌体的能力取决于细胞环境和生理状态。这种能力可以在不同条件下产生动态变化。

应当强调的是，外泌体的一个重要的功能就是介导细胞间的相互作用：在肿瘤微环境中，癌细胞释放的外泌体可被基质细胞吸收，从而将微环境转化为适宜肿瘤生长的状态；另一方面，基质细胞也可以释放外泌体被癌细胞吸收并促进肿瘤生长。由于癌细胞的异质性，某些肿瘤细胞分泌的外泌体还可以介导与其相邻的肿瘤细胞的基因信息交换，以改变它们的生物表型，如诱导耐药性^[11-12]。

受体细胞是如何摄取外泌体的并且受体细胞是如何被选择的是件有趣的事情。外泌体具有细胞特异性；然而，受体细胞被选择，机制尚不十分清楚。有几篇报告指出，这可能与外泌体表面的粘附相关分子有关，如tetrapanins，糖蛋白，整合素和SNAREs^[13-14]。最近David Lyden博士的研究组发表于Nature的文献表明，乳腺癌细胞分泌的外泌体具有独特的整合素表达模式^[6]。带有α6β4和α6β1整合素的外泌体可被肺中的S100A4阳性的成纤维细胞和表面活性蛋白C（SPC）阳性的上皮细胞获取，从而促进肺转移；带有αVβ5整合素的外泌体被位于肝脏中的kupffer细胞有选择地摄取，从而促进肝转移。因此，整合素分子在外泌体转移和吸收过程中起着至关重要的作用。

生物谷：关于外泌体的应用开发方面，您认为外泌体在疾病的诊断和治疗方面哪个更有应用前景？各应用方向有哪些发展的瓶颈问题？

回答：外泌体的重要应用包括利用外泌体作为生物标志物用于疾病诊断、预后和治疗手段。虽然外泌体作为生物标志物用于诊断或预后在转化应用上推进很快，但基于外泌体的癌症诊断和治疗两方面都有很好的前景。外泌体可以在诸如血，尿，唾液和脑脊液等体液中检测到。因此，它所携带的分子是理想的非侵入性肿瘤诊断标记物。

一个很好的例子是，研究发现胰腺癌外泌体表面富含蛋白聚糖，磷脂酰肌醇聚糖-1（glypican-1， GPC1），它可以用作胰腺癌的一个高灵敏度和特异性的生物标志物^[15]。虽然GPC1作为胰腺癌诊断的生物标志物仍需要在更大的患者群进行验证，但这一发现展示了外泌体诊断癌症的强大力量。

使用外泌体诊断癌症的主要挑战是：1）灵敏度和特异性。不是每一个外泌体蛋白质生物标志物都像GPC1那样灵敏度高和特异性好。许多候选生物标志物的灵敏度和特异性并不比目前的癌症生物标志物更好，他们大多数还没有表现出明显的临床应用的希望。2）从血液或其它体液中分离和富集外泌体的技术方面仍需进一步改进。

在回答前一个问题时，我们已经讨论了外泌体在癌症治疗中的作用。外泌体用于癌症治疗的主要挑战包括但不仅限于，1）如何将外源性的miRNAs，siRNAs或药物装进外泌体，如何促进其被特定细胞吸收；2）在开发非自体同源的外泌体或生物工程化的外泌体时，如何防止其引起炎症和免疫反应； 3）如何提高外泌体在体内的半衰期，并避免注射后被肝和肾快速清除；4）从病人血液中去除肿瘤来源的外泌体时，如何同时保留具有正常生理功能对肿瘤无促进作用的外泌体。5）用于患者治疗的大量外泌体的分离的成本和技术挑战。外泌体同时参与促肿瘤和抗肿瘤两种功能。

因此，在临床试验中广泛推行外泌体之前，我们应当彻底了解外泌体的生物复杂性。

生物谷：我们目前对于外泌体的分离提取手段并不能得到100% pure的外泌体，您怎么看待这个问题，这影响我们对外泌体的研究和应用开发吗？

回答：是的，当前的技术并不能得到100％纯的外泌体，这可能在外泌体的临床应用上造成负面影响。通常，外泌体是通过超速离心或亲和珠分离的，并通过它们表面标志物和密度特异性进一步富集外泌体。因此，这些方法的制备外泌体可能含有其他小的细胞外膜泡或其他成分，而不是"百分之百的"外泌体。

为了更好地从其他细胞外囊泡中分离出外泌体，Clotilde Thery博士的研究组^[8,16]和其他团队在规范外泌体纯化方法和外泌体的定义方面做了大量的工作。外泌体纯化方法的规范和技术的完善可以帮助生产更纯的外泌体，提高实验数据的可靠性和可重复性，增加临床应用外泌体的质量保证。我们预计这一令人兴奋的领域将会迅速发展和进步。

余棣华 M.D.，PHD.

美国德州大学MD安德森癌症中心

余棣华，毕业于首都医科大学，美国德州大学MD安德森癌症中心终身教授。 余棣华教授早年毕业于首都医科大学，是我国第一代出国深造的海外留学生。余棣华教授于1982年毕业于首都医科大学，获医学学士学位。1985年获首都医科大学生理学硕士学位。1986年后赴美学习，1991年于University of Texas 获博士学位(Ph.D.)。现为美国德州大学MD安德森癌症中心终身教授，Nylene Eckles 杰出教授，Hubert L. & Olive Stringer杰出教授，外科肿瘤实验室主任，肿瘤分子细胞学系副主任。

以下是英文采访原文，供参考：

生物谷： 关于您Nature文章：脑微环境中存在如此促进肿瘤定植的因子,您认为会不会是一种普遍现象,是否可能存在很多这种类似的miR。肿瘤一旦突破血脑屏障就不再受抑制？

回答： Our studies showed that astrocytes-derived miR-19a inhibited PTEN expression in metastatic tumor cells, thereby priming brain metastasis outgrowth. This mechanism is shared by breast cancer and melanoma brain metastasis models. miR-19a is one of the microRNAs that down regulate PTEN in brain metastatic cells, but we expect that some other PTEN-targeting microRNAs derived from astrocytes might have a similar function as miR-19a. This mechanism of astrocytes-derived miRs controlling the gene expression of disseminate tumor cells might also be involved in PTEN loss in other neurologic disease. Once disseminate tumor cells pass through the blood-brain barrier, the brain microenvironment could elicit both inhibiting and promoting effects on tumor colonization and outgrowth. Brain metastasis can have a long dormancy before it relapses, implying that the dynamic crosstalk between the disseminate tumor cells and brain metastatic niches is required for successful tumor colonization and outgrowth. Once disseminate tumor cells succeed in negotiating with brain microenvironment, and reversely the brain microenvironment shows the welcome signal to tumor cells, metastatic tumors would outgrow to symptomatic diseases.

生物谷：您怎么看外泌体在肿瘤治疗方面的应用？

回答：Exosomes represent a type of bio-vehicles that could be considered as natural liposomes. They are membrane-permeable, well-tolerated and can cross the blood-brain barrier. Therefore, exosomes emerge as favorable candidate carriers for therapeutics of cancer and other diseases. The first appealing application of exosome is the delivery of drugs, microRNAs, siRNAs and other therapeutic compounds, because they are more stable in exosomes ^[1]. For example, exosomes could be used to deliver tumor suppressor microRNAs or siRNAs that knock down oncogenes to inhibit tumor growth. Exosomes could target specific cell types or tissues so exosome delivery may improve efficacy and reduce off-target effects. Synthetic target ligand expression on the surface of exosomes can further improve their cell or tissue targeting specificity. For example, exosomes were engineered to express Lamp2b with a tumor-targeting integrin to enhance tumor-specific uptake ^[2]. Secondly, exosomes can be applied for cancer vaccination. Exosome can deliver tumor-derived antigens to induce anti-tumor immune responses. Dendritic cell-derived exosomes can induce anti-tumor immunity and have been used in clinical trials ^[3-5]. Thirdly, considering cancer-cell derived exosome can modify pre-metastasis niches ^[6], depleting these exosomes from circulation system is a possible option for blocking cancer metastasis. Reducing exosomes in the circulation system can be achieved by inhibiting exosome assembly and release from tumor cells with inhibitors, or by eliminating exosomes from cancer patients' circulation via extracorporeal purification. In summary, we can be optimistic about using exosomes as a new venue for cancer therapy despite of the technical challenges. Many investigators have shown evidences supporting the application of exosome for cancer treatment in vitro and in vivo ^[7]. However, currently only a few clinical trials using exosomes in cancer therapy are implemented. Future studies should clarify the complex mechanism of exosomes for more efficacious clinical trials.

生物谷：分泌外泌体的主细胞和其他细胞都可以吞噬或者接受外泌体携载的活性分子如mRNA,miRNA等,主细胞与其他细胞对外泌体摄取或者结合哪个更有优势？可能机制是什么？

回答： Exosomes are secreted by nearly all mammalian cells in the body, and also many cells can uptake exosomes containing mRNA, miRNA, or others. Cancer cells, fibroblast cells, immune cells, astrocytes and other cells have all been reported to release or uptake exosomes ^[7-9]. Indeed, donor cells can re-uptake their secreted exosomes. For example exosomes produced by pancreatic tumor cell lines had an autocrine effect on themselves shown by in vitro experiments^[10]. However, the more important role of exosome seems to be modulation of microenvironment by paracrine mechanisms. Those exosomes from host cells can serve as external stimuli for recipient cells and change recipient cell signaling as well as the microenvironment around host cells. It is conceivable that the ability of up-taking exosomes by host cells themselves or by recipient cells depends on cellular context, and physiological status. This ability could dynamically change under different conditions. It should be emphasized that a significant function of exosomes is mediating intercellular interactions： cancer cell-released exosomes can be taken up by stromal cells in the tumor microenvironment that can thus be converted to tumor-prone microenvironment； reciprocally stromal cells in the tumor microenvironment can release exosomes taken up by cancer cells and facilitate tumor growth. Due to the heterogeneous nature of cancer cells, exosomes secreted by some tumor cells could also mediate the exchange of gene information to their neighboring tumor cells to alter their biological phenotypes, such as inducing drug resistance ^[11-12].

It is intriguing how the recipient cells uptake exosomes and how recipient cells are chosen. Exosomes are cell specific； however the mechanism of how recipient cells be selected is not yet very clear. Several reports indicated that it might be determined by adhesion-associated molecules on the surface of exosome, such as tetrapanins, glycoproteins, integrins and SNAREs ^[13-14]. A recent amazing Nature paper from Dr. David Lyden's group ^[6] showed that exosomes released by breast cancer cells had distinct integrin expressions. Exosomes having α6β4 and α6β1 integrins can be up-taken by S100A4-positive fibroblasts and surfactant protein C (SPC)-positive epithelial cells in the lungs to facilitate lung metastasis； exosomes with αvβ5 integrins is selectively uptaken by kupffer cells in the liver to promote liver metastasis. Therefore, integrins play critical roles in governing exosome transfer and uptake.

生物谷：关于外泌体的应用开发方面,您认为外泌体在疾病的诊断和治疗方面哪个更有应用前景？各应用方向有哪些发展的瓶颈问题？

回答：Significant applications of exosome include using exosomes as biomarkers for disease diagnosis, prognosis and as therapeutics. Although exosome biomarkers for diagnosis or prognosis seem to be on the fast track for translational application, both cancer diagnosis and therapy based by exosomes have bright perspectives. Exosomes can be detected in the body fluid, such as blood, urine, saliva and cerebrospinal fluid. Therefore, it is an ideal non-invasive biomarker for tumor diagnosis. A great example is that exosomes from pancreatic cancers were found to be enriched with the cell surface proteoglycan, glypican-1 (GPC1) which can be used as a highly sensitive and specific biomarker of pancreatic cancer ^[15]. Although GPC1 as a biomarker of pancreatic cancer need to be validated in larger patient cohorts, this finding exemplifies the power of utilizing exosomes for cancer diagnose. The main challenges of using exosomes in cancer diagnosis are 1) sensitivity and specificity. Not every exosomal proteins are biomarkers as sensitive and specific as described for GPC1. Many biomarker candidates' sensitivity and specificity are no better than current cancer biomarkers and most of them have not shown a clear promise for clinical application. 2) The technical aspects for isolating and enriching exosomes from the blood or other bodily fluids need to be further improved.

We have already discussed the role of exosome in cancer therapy when answering the previous question. The main challenges of using exosomes in cancer therapy include, but not limited to, 1) How to load exogenous miRNAs, siRNAs or drugs into exosomes and how to increase cell-specific transfer； 2) How to prevent inflammation and immune reactions when developing non-autologous exosomes or bioengineered exosomes； 3) How to increase the half-life of exosomes in the body and avoid rapid clearance by liver and kidney after injection； 4) When depleting exosomes in the blood of patients, how to prevent loss of normal physiological functions of non-tumor promoting exosomes. 5) The cost concern and technology challenges related to isolating large amounts of exosomes for the treatment of patients. Exosomes can partake in both pro- and anti-tumor functions. Therefore, we should thoroughly understand the complexity of exosome biology before widely implementing exosomes in clinical trials.

生物谷：我们目前对于外泌体的分离提取手段并不能得到100% pure的外泌体,您怎么看待这个问题,这影响我们对外泌体的研究和应用开发吗？

回答：Yes, the current technologies do not yield 100% pure exosomes which could negatively impact on the exosomes' clinical application. Frequently, exosomes are isolated by centrifugation or by affinity beads, and exosomes are enriched by their surface markers and density. Consequently, these exosome preparations may contain other small extra-celluar vesicles or other components, rather than "true" exosomes. To better separate exosomes from other extracelluar vesicles, Dr. Clotilde Thery's group ^{[8, 16]} and other teams devoted efforts to standardize exosome purification methods and the definition of exosomes. Standardization of exosome purification protocol, along with technology improvements can help to produce more pure exosomes, improve the reliability and reproducibility of experimental data, and increase the quality assurance of exosome for clinical applications. We anticipate that this exciting field will be rapidly evolving and progressing.

Reference：

1.Johnsen, KB. et al. A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy. (Review) Biochum Biophys Acta 1846, 75-75 (2014).

2.Tian, Y. et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biometerials 35, 2383-2390 (2014).

3.Escudier, B. et al. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes： results of the first phase I clinical trial. J Transl Med. 3, 1-13 (2005).

4.Morse, MA. et al. A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. J Transl Med. 3, 9 (2005).

5.Dai, S. et al. Phase I clinical trial of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer. Mol Ther. 16, 782-790 (2008).

6.Hoshina, A. et al. Tumour exosome integrins determine organotropic metastasis. Nature 527, 329-335 (2015).

7.Munson, P. et al. Exosomes： Potential in cancer diagnosis and therapy. (Review) Medicine 2, 310-327 (2015).

8.Kowal, J. et al. Biogenesis and secretion of exosomes. Curr Opin Cell Biol. 29, 116-125 (2014).

9.Zhang, L. et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 527, 100-104 (2015).

10.Ristorcelli E. et al. Essential role of Notch signaling in apoptosis of human pancreatic tumoral cells mediated by exosomal nanoparticles. Int J Cancer. 125, 1016-1026 (2009).

11.Bebawy, M. et al. Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells. Leukemia 23, 1643-1649 (2009).

12.Corcoran, C. et al. Docetaxel-resistance in prostate cancer： evaluating associated phenotypic changes and potential for resistance transfer via exosomes. PLoS One 7, e50999 (2012).

13.Escrevente, C. et al. Interaction and uptake of exosomes by ovarian cancer cells. BMC Cancer 11, 108 (2011).

14.Rana, S. et al. Toward tailored exosomes： the exosomal tetraspanin web contributes to target cell selection. Int J Biochem Cell Biol. 44, 1574-1584 (2012).

15.Mela, SA. et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523, 177-182 (2015).

16.Colombo, M. et al. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 30, 255-289 (2014).

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