国家癌症中心和耶鲁大学: 发现肿瘤细胞成群结队聚集转移
Targeting clusters of circulating tumor cells to suppress metastasis
要点：
1. 癌细胞转移是一个非常低效的过程；大的肿瘤每天释放高达100万个的肿瘤细胞，进入血液，只有少数几个能存活定居下来；
2. 癌细胞的转移必须通过血液循环，通常需在血流中存留3天；（提示健康的血液有大量能识别和杀死癌细胞免疫细胞，就能歼灭血循环中的癌细胞）；
3. 癌细胞常常聚集在一起，成群结队的旅行-这是少数癌细胞能下血液这个非常不利的环境中存活下来的关键；
4. 癌细胞聚集（细胞-细胞连结）在一起依赖于黏蛋白，而黏蛋白依赖于钙离子的存在；镁离子是钙离子的天然拮抗物。这个关联是富含镁离子的蔬菜汁有抗癌功效的依据之一。
图:从乳腺癌患者血液中分离出来的循环肿瘤细胞簇的代表性图像，用广谱细胞角蛋白(红色)和DAPI(蓝色细胞核)染色。
最近，突破性的技术进步首次使从癌症患者的血液中分离和鉴定异常罕见的循环肿瘤细胞成为可能。使用患者样本和小鼠模型，结合微流体技术和单细胞解析RNA测序，我们发现循环肿瘤细胞簇（CTC-Cluster），由强大的细胞连接连接在一起，代表癌症患者循环中的转移前体。此外，我们还开发了一种新的微流体技术，能够从癌症患者未经处理的血液中分离出CTC簇。总之，这些研究强调了CTC-cluster和细胞-细胞连接是一种以前未被认识和潜在靶向的癌症传播机制。
癌症转移是绝大多数癌症相关死亡的原因，目前还没有有效的治疗方法来抑制转移性疾病。
了解患者癌症转移发生的复杂而基本的机制对于开发成功的治疗方法至关重要。虽然我们对转移发生机制的大部分理解都来自于小鼠模型，但对癌症患者血液循环中人类转移前体的分析受到技术限制的极大限制。最近，突破性的技术进步使得首次从转移性癌症患者的血液中分离和查询罕见循环肿瘤细胞成为可能，揭示了转移过程中意想不到的特征。
图:从乳腺癌患者血液中分离出来的循环肿瘤细胞簇的代表性图像，用广谱细胞角蛋白(红色)和DAPI(蓝色细胞核)染色。
我们发现，尽管患者中大多数循环肿瘤细胞（CTC）是单细胞的，但罕见的CTC簇(CTC-cluster)(见图)，比单个CTC突破和转移的有效率提高50倍。CTC簇是通过表达细胞-细胞连接成分(如plakoglobin. 一种细胞粘蛋白)而聚集在一起的。在乳腺癌患者原发肿瘤中，plakoglobin蛋白的表达导致无进展生存率降低。此外，在小鼠模型中靶向plakoglobin抑制了CTC簇的形成和转移。
基于发现CTC簇的高度转移倾向及其对细胞-细胞连接的依赖性，我们开发了一种新的微流体技术(簇状芯片)，使CTC簇能够从未经处理的人体血液样本[2]中分离和表征。在低剪切应力条件下，簇状芯片通过专门的分叉圈闭分离CTC簇。利用这项技术，我们从乳腺癌、前列腺癌和黑色素瘤患者中分离出CTC簇，并进一步鉴定了与CTC簇相关的免疫系统成分。
综上所述，这些结果表明细胞-细胞连接和CTC聚类是癌症传播的基本机制，值得进一步研究，以开发针对癌症患者的转移特异性疗法。
Targeting clusters of circulating tumor cells to suppress metastasis
Recently, breakthrough technological advances have made it possible, for the first time, to isolate and characterize extraordinarily rare circulating tumor cells (CTCs) from the blood of patients with cancer. Using both patient samples and mouse models, coupled with microfluidics technology and single cell resolution RNA sequencing, we found that CTC-clusters, held together by strong cell-cell junctions, represent metastatic precursors in the circulation of patients with cancer. Furthermore, we developed a new microfluidics technology capable of isolating CTC-clusters from the unprocessed blood of patients with cancer. All together, these studies highlight CTC-clusters and cell-cell junctions as a previously unappreciated and potentially targetable mechanism of cancer dissemination.
HFSP Long-Term Fellow Nicola Aceto and colleagues
authored on Tue, 26 May 2015
Cancer metastasis accounts for the vast majority of cancer-related deaths, and, currently, no treatments are available to suppress a metastatic disease. Understanding the complex, yet fundamental mechanisms underlying the development of cancer metastasis in patients is critical for developing successful therapies. While most of our understanding of how metastasis occurs is derived from mouse models, the analysis of human metastatic precursors in the circulation of patients with cancer has been greatly limited by technological constraints. Recently, breakthrough technological advances have made it possible to isolate and interrogate for the first time rare circulating tumor cells (CTCs) from the blood of patients with metastatic cancers, revealing unexpected features of the metastatic process.
Figure: Representative image of a circulating tumor cell cluster isolated from the blood of a patient with breast cancer and stained with wide-spectrum cytokeratin (red) and DAPI (nuclei, blue).
We found that, while the majority of CTCs in patients are found as single cells, rare clusters of CTCs (CTC-clusters) (see figure) can break off the primary tumor and metastasize up to 50 times more efficiently than single CTCs [1]. CTC-clusters are held together by the expression of cell-cell junction components such as plakoglobin. Expression of plakoglobin in the primary tumor of breast cancer patients results in a reduced progression-free survival. Furthermore, targeting plakoglobin in mouse models suppresses the formation of CTC-clusters and metastasis.
Based on the findings that revealed the high metastatic propensity of CTC-clusters and their dependence upon cell-cell junctions, we developed a new microfluidics technology (the Cluster-Chip) to enable the isolation and characterization of CTC-clusters from unprocessed human blood samples [2]. The Cluster-Chip isolates CTC-clusters through specialized bifurcating traps under low shear stress conditions. With this technology, we isolated CTC-clusters from patients with cancers of the breast, prostate and melanoma, and further characterized immune system components associated with CTC-clusters.
All together, these results suggest cell-cell junctions and CTC-clustering as a fundamental mechanism of cancer dissemination, and warrant further studies to enable the development of metastasis-tailored therapies for patients with cancer.
References
[1] Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, Yu M, Pely A, Engstrom A, Zhu H, Brannigan BW, Kapur R, Stott SL, Shioda T, Ramaswamy S, Ting DT, Lin CP, Toner M, Haber DA and Maheswaran S. Cell. 2014 Aug 28;158(5):1110-22. PMID: 25171411
[2] Microchip technology for antigen-independent isolation of circulating tumor cell clusters. Sarioglu FA*, Aceto N*, Kojic N, Donaldson MC, Zeinali M, Hamza B, Engstrom A, Zhu H, Sundaresan T, Miyamoto DT, Luo X, Bardia A, Wittner BS, Ramaswamy S, Shioda T, Ting DT, Stott SL, Kapur R, Maheswaran S, Haber DA and Toner M. *Equal contribution.Nature Methods. 2015 May 18. doi: 10.1038/nmeth.3404. PMID: 25984697
Targeting clusters of circulating tumor cells to suppress metastasis | Human Frontier Science Program
2. https://link.springer.com/article/10.1007%2Fs10549-015-3416-1
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