在台灣，肺癌的發生率僅次於肝癌，在癌症相關之致死率則是位於第一名，其中高達九成造成癌症病患死亡的原因都是因為癌轉移，而在臨床上肺癌最常發生轉移之器官包含了大腦、骨頭、肝臟等等，依照目前的醫學研究在防止癌轉移的發生上仍舊有難度。但近年來研究發現，癌細胞會相較於正常細胞製造大量的胞外泌體去做為細胞與細胞溝通的橋樑，透過運送活性因子起始癌轉移的訊息傳遞、改變腫瘤的微環境、甚至能為癌細胞預備轉移前的生長環境，促使癌細胞轉移，然而在胞外泌體之蛋白所調控的轉移機制目前仍不清楚。在實驗室先前之研究中已發現，肺腺癌細胞低侵襲性的 CL1-0 在接受高侵襲性 CL1-5 的胞外泌體後，會增加其細胞爬行的能力。因此本篇研究假設 CL1-5 所分泌的胞外泌體中，包裹著特定蛋白可以影響較不惡性的 CL1-0 細胞並增加移動能力最終達到癌轉移。
首先在動物實驗結果中，可以發現高侵襲性的CL1-5確實會促進低侵襲性的CL1-0癌轉移之特性。為了著重在胞外泌體調控的機制，我們利用 GFP-CD9 標定 CL1-5 的胞外泌體，並利用共培養以及Transwell 系統在體外觀察到CL1-0確實會內吞來自於CL1-5的胞外泌體並增加其爬行之能力。進一步以蛋白質體分析探討 CL1-5 的胞外泌體中是否含有某特定蛋白可調控 CL1-0 的爬行能力，在結果中所找到的兩個候選蛋白 Ephrin receptor A2 (EPHA2) 和 EH-domain containing protein1 (EHD1) 確實在 CL1-5 細胞及胞外泌體中都具有較高表現的。最後我們在CL1-5細胞中減弱EPHA2及EHD1時，確實會抑制細胞之爬行能力，並且此現象是透過影響Src以及Akt的蛋白磷酸化及其下游路徑所造成的。
總結來看本研究發現來自高侵襲性的CL1-5胞外泌體中的特定的蛋白EPHA2及EHD1會傳送至低侵襲性的非小細胞肺癌，並且促進其爬行能力，最終達到癌轉移的現象發生。此胞外泌體所含有的蛋白可做為預測或是預後的生物標記，用於早期診斷或是治療預後的工具，若更進一步可以利用藥物或是標靶治療抑制胞外泌體，或許在臨床上可作為一個新的治療方針。

In Taiwan, lung cancer is now the second most common cancer only less than liver cancer and has the highest mortality. Cancer metastasis is responsible for 90% of the cancer-related death. In clinical, the brain, bone, and liver are the most metastatic sites of lung cancer. It’s still difficult to stop the happen of cancer metastasis because its underlying mechanism is still unclear. Recently, exosomes derived from tumor cells have been reported that could regulate cell-to-cell communication. Exosomes-carried bioactive molecules also have been demonstrated that could initiate the cancer metastasis cascade, modulate the tumor microenvironment and prepare the “pre-metastatic niche” for cancer metastasis. However, the mechanism of tumor-derived exosomal proteins-mediated cancer metastasis in lung cancer is still unclear. In our previous data indicated that the low-invasive lung adenocarcinoma CL1-0 cells could receive the exosomes from highly invasive CL1-5 cells and increase the cell migration ability. Here, we hypothesize that CL1-5 cells release the exosomal proteins that transfer to CL1-0 cells and promote the aggressiveness of CL1-0 cells then initiate the cancer metastasis.
In animal models, the results showed that CL1-5 promoted CL1-0 cells more aggressive in vivo. To focus on the mechanism of exosome-mediated cancer promotion we labeled CL1-5 cells with GFP-CD9 and co-cultured the cells with CL1-0 cells in a transwell system. The data indicated that the CL1-5-released exosomes were internalized into CL1-0 and further enhanced CL1-0 cells migration ability. Moreover, the exosomal proteins derived from CL1-0 and CL1-5 were identified by LC-MS/MS. The analysis identified that EHD1 and EPHA2 were highly expressed in CL1-5 cellular and exosomal proteins compared with CL1-0. After silencing the EPHA2 or EHD1 in CL1-5 cells, the migration ability and the phosphorylation levels of Akt and Src of CL1-5 cells were significantly decreased. These results suggested that EHPA2 and EHD1 might involve in exosome-dependent CL1-5-promoted cell migration of CL1-0 cells.
According to these data, we demonstrated that the EPHA2 and EHD1, which were in the exosomes derived from highly invasive CL1-5 cells, are transferred into low-invasive CL1-0 cells to enhance cell migration and then promote cancer metastasis. The exosomal proteins could be used as the biomarkers for prediction or prognosis of cancer. Moreover, blocking the exosomes might be served as the therapeutic strategy for lung cancer metastasis.

1 Amos, C. I. et al. P1.04: Defining the Genetic Architecture of Lung Cancer Etiology. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 11, S182-S182, doi:10.1016/j.jtho.2016.08.026 (2016).
2 Herbst, R. S., Morgensztern, D. & Boshoff, C. The biology and management of non-small cell lung cancer. Nature 553, 446, doi:10.1038/nature25183
https://www.nature.com/articles/nature25183#supplementary-information (2018).
3 Ho, Y. R., Ma, S.-P. & Chang, K. Y. Trends in regional cancer mortality in Taiwan 1992–2014. Cancer Epidemiology 59, 185-192, doi:https://doi.org/10.1016/j.canep.2019.02.005 (2019).
4 Tamura, T. et al. Specific organ metastases and survival in metastatic non-small-cell lung cancer. Mol Clin Oncol 3, 217-221, doi:10.3892/mco.2014.410 (2015).
5 Chaffer, C. L. & Weinberg, R. A. A perspective on cancer cell metastasis. science 331, 1559-1564 (2011).
6 Bravo-Cordero, J. J., Hodgson, L. & Condeelis, J. Directed cell invasion and migration during metastasis. Current Opinion in Cell Biology 24, 277-283, doi:https://doi.org/10.1016/j.ceb.2011.12.004 (2012).
7 Ridley, A. J. et al. Cell migration: integrating signals from front to back. Science 302, 1704-1709 (2003).
8 Fumarola, C., Bonelli, M. A., Petronini, P. G. & Alfieri, R. R. Targeting PI3K/AKT/mTOR pathway in non small cell lung cancer. Biochemical pharmacology 90, 197-207 (2014).
9 Liu, L. & Parent, C. A. TOR kinase complexes and cell migration. J Cell Biol 194, 815-824 (2011).
10 Gu, K. et al. Interleukin-17-induced EMT promotes lung cancer cell migration and invasion via NF-kappaB/ZEB1 signal pathway. American journal of cancer research 5, 1169-1179 (2015).
11 Brábek, J., Mierke, C. T., Rösel, D., Veselý, P. & Fabry, B. The role of the tissue microenvironment in the regulation of cancer cell motility and invasion. Cell communication and signaling 8, 22 (2010).
12 Tetta, C., Ghigo, E., Silengo, L., Deregibus, M. C. & Camussi, G. Extracellular vesicles as an emerging mechanism of cell-to-cell communication. Endocrine 44, 11-19 (2013).
13 Wolf, P. The nature and significance of platelet products in human plasma. British journal of haematology 13, 269-288 (1967).
14 Dvorak, H. F. et al. Tumor shedding and coagulation. Science 212, 923-924 (1981).
15 Dvorak, H. F. et al. Procoagulant activity associated with plasma membrane vesicles shed by cultured tumor cells. Cancer Research 43, 4434-4442 (1983).
16 Xu, R. et al. Extracellular vesicles in cancer - implications for future improvements in cancer care. Nature reviews. Clinical oncology 15, 617-638, doi:10.1038/s41571-018-0036-9 (2018).
17 Maas, S. L., Breakefield, X. O. & Weaver, A. M. Extracellular vesicles: unique intercellular delivery vehicles. Trends in cell biology 27, 172-188 (2017).
18 Raposo, G. & Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200, 373-383, doi:10.1083/jcb.201211138 (2013).
19 Suetsugu, A. et al. Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude-mouse models. Advanced drug delivery reviews 65, 383-390 (2013).
20 Becker, A. et al. Extracellular vesicles in cancer: cell-to-cell mediators of metastasis. Cancer cell 30, 836-848 (2016).
21 Meehan, K. & Vella, L. J. The contribution of tumour-derived exosomes to the hallmarks of cancer. Critical reviews in clinical laboratory sciences 53, 121-131 (2016).
22 Ruivo, C. F., Adem, B., Silva, M. & Melo, S. A. The biology of cancer exosomes: insights and new perspectives. Cancer research 77, 6480-6488 (2017).
23 Richards, K. E. et al. Cancer-associated fibroblast exosomes regulate survival and proliferation of pancreatic cancer cells. Oncogene 36, 1770 (2017).
24 Singh, R., Pochampally, R., Watabe, K., Lu, Z. & Mo, Y.-Y. Exosome-mediated transfer of miR-10b promotes cell invasion in breast cancer. Molecular cancer 13, 256 (2014).
25 Raimondo, S. et al. Chronic myeloid leukemia-derived exosomes promote tumor growth through an autocrine mechanism. Cell Communication and Signaling 13, 8 (2015).
26 Shan, Y. et al. Hypoxia-induced matrix Metalloproteinase-13 expression in exosomes from nasopharyngeal carcinoma enhances metastases. Cell death & disease 9, 382 (2018).
27 You, Y. et al. Matrix metalloproteinase 13-containing exosomes promote nasopharyngeal carcinoma metastasis. Cancer science 106, 1669-1677, doi:10.1111/cas.12818 (2015).
28 Luga, V. et al. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 151, 1542-1556 (2012).
29 Webber, J., Steadman, R., Mason, M. D., Tabi, Z. & Clayton, A. Cancer exosomes trigger fibroblast to myofibroblast differentiation. Cancer research 70, 9621-9630 (2010).
30 Whiteside, T. L. (Portland Press Limited, 2013).
31 Chow, A. et al. Macrophage immunomodulation by breast cancer-derived exosomes requires Toll-like receptor 2-mediated activation of NF-κB. Scientific reports 4, 5750 (2014).
32 Whiteside, T. L. in Advances in Clinical Chemistry Vol. 74 (ed Gregory S. Makowski) 103-141 (Elsevier, 2016).
33 Paget, S. The distribution of secondary growths in cancer of the breast. The Lancet 133, 571-573 (1889).
34 Tai, Y. L., Chen, K. C., Hsieh, J. T. & Shen, T. L. Exosomes in cancer development and clinical applications. Cancer science 109, 2364-2374 (2018).
35 Chen, I. H. et al. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. Proceedings of the National Academy of Sciences of the United States of America 114, 3175-3180, doi:10.1073/pnas.1618088114 (2017).
36 Chu, Y.-W. et al. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. American journal of respiratory cell and molecular biology 17, 353-360 (1997).
37 Meng, Q. et al. Mammalian Eps15 homology domain 1 promotes metastasis in non-small cell lung cancer by inducing epithelial-mesenchymal transition. Oncotarget 8, 22433 (2017).
38 Teng, Y. et al. MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression. Nat Commun 8, 14448, doi:10.1038/ncomms14448 (2017).
39 Hoshino, A. et al. Tumour exosome integrins determine organotropic metastasis. Nature 527, 329-335, doi:10.1038/nature15756 (2015).
40 Meyer-Schaller, N., Heck, C., Tiede, S., Yilmaz, M. & Christofori, G. Foxf2 plays a dual role during transforming growth factor beta-induced epithelial to mesenchymal transition by promoting apoptosis yet enabling cell junction dissolution and migration. Breast Cancer Research 20, 118, doi:10.1186/s13058-018-1043-6 (2018).
41 Le, M. T. N. et al. miR-200–containing extracellular vesicles promote breast cancer cell metastasis. The Journal of Clinical Investigation 124, 5109-5128, doi:10.1172/JCI75695 (2014).
42 Boelens, M. C. et al. Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways. Cell 159, 499-513, doi:10.1016/j.cell.2014.09.051 (2014).
43 Leca, J. et al. Cancer-associated fibroblast-derived annexin A6+ extracellular vesicles support pancreatic cancer aggressiveness. The Journal of Clinical Investigation 126, 4140-4156, doi:10.1172/JCI87734 (2016).
44 Thayanithy, V. et al. A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication. Cell communication and signaling : CCS 15, 46, doi:10.1186/s12964-017-0201-2 (2017).
45 Han, J.-Y. et al. Immunohistochemical expression of integrins and extracellular matrix proteins in non-small cell lung cancer: correlation with lymph node metastasis. Lung Cancer 41, 65-70 (2003).
46 Kapil, S. et al. The cell polarity protein Scrib functions as a tumor suppressor in liver cancer. Oncotarget 8, 26515 (2017).
47 Xia, Z. et al. Nonmuscle myosin IIA is associated with poor prognosis of esophageal squamous cancer. Diseases of the Esophagus 25, 427-436 (2012).
48 Xiong, D. et al. Non-muscle myosin II is an independent predictor of overall survival for cystectomy candidates with early-stage bladder cancer. Oncology reports 28, 1625-1632 (2012).
49 Liu, D. et al. Clinicopathological Significance of NMIIA Overexpression in Human Gastric Cancer. International journal of molecular sciences 13, 15291-15304 (2012).
50 Katono, K. et al. Prognostic significance of MYH9 expression in resected non-small cell lung cancer. PLoS One 10, e0121460 (2015).
51 Cai, B. et al. Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes. The Journal of biological chemistry 288, 30172-30180, doi:10.1074/jbc.M113.488627 (2013).
52 Naslavsky, N., Boehm, M., Backlund, P. S., Jr. & Caplan, S. Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane. Molecular biology of the cell 15, 2410-2422, doi:10.1091/mbc.e03-10-0733 (2004).
53 Mathivanan, S., Ji, H. & Simpson, R. J. Exosomes: Extracellular organelles important in intercellular communication. Journal of Proteomics 73, 1907-1920, doi:https://doi.org/10.1016/j.jprot.2010.06.006 (2010).
54 Wykosky, J. & Debinski, W. The EphA2 Receptor and EphrinA1 Ligand in Solid Tumors: Function and Therapeutic Targeting. Molecular Cancer Research 6, 1795, doi:10.1158/1541-7786.MCR-08-0244 (2008).
55 Huang, J. et al. EphA2 promotes epithelial–mesenchymal transition through the Wnt/β-catenin pathway in gastric cancer cells. Oncogene 33, 2737, doi:10.1038/onc.2013.238
https://www.nature.com/articles/onc2013238#supplementary-information (2013).
56 Miyazaki, T., Kato, H., Fukuchi, M., Nakajima, M. & Kuwano, H. EphA2 overexpression correlates with poor prognosis in esophageal squamous cell carcinoma. International journal of cancer 103, 657-663, doi:10.1002/ijc.10860 (2003).