臺灣博碩士論文加值系統

肺癌的新病例及死亡人數皆高居癌症首位，易轉移與復發的預後被認為與癌幹細胞（Cancer stem cell, CSC）有密切關連。癌幹細胞具有強力的自我更新、侵襲、腫瘤生成和抗藥性，能抵抗藥物毒殺而導致腫瘤復發及轉移，因此探討如何抑制癌幹細胞生成及特性是治療的重要議題。近年來，研究發現趨化因子及其受體（Chemokine and chemokine receptor）在腫瘤微環境中的表現異常與癌症進程有高度相關，然而成員及功能的複雜性使其在肺癌進程中所扮演的角色至今仍尚未被完全了解。
　文獻指出，黑色素瘤、膠質母細胞瘤及腎臟癌中CCR3表現量與癌症惡性程度呈正相關，且CCL7及CCL11會經由CCR3而促進細胞增生、移動及腫瘤生長。臨床研究顯示，大腸直腸癌病患血清中CCL13的表現可作為遠端轉移的指標，先前實驗室透過細胞激素微陣列分析肺癌腫瘤微球體（Tumor sphere）發現，CCR3的配體（Ligand）──CCL13之表現量有大幅地上升。到目前為止，CCR3與CCL13在肺癌進程和癌幹細胞都尚未有相關研究，因此本論文旨在探討CCR3與CCL13在肺癌進程及癌幹細胞中所扮演的角色。
　本論文以兩種不同惡性程度的肺腺癌細胞A549與H1299作為實驗模組，結果發現不論是短期處理轉化生長因子-b1（Transforming growth factor-b1）或培養成腫瘤微球體，CCR3與CCL13的基因表現量皆大幅上升，更加確認CCR3/CCL13軸在肺癌進程及癌幹細胞中的重要性。以慢病毒短髮夾核糖核酸系統（Lentivirus shRNA system）抑制CCR3的表現（shCCR3），發現癌細胞的生長及移動能力皆受到抑制，並且可抑制由TGF-b1介導的間質樣（Mesenchymal-like）細胞型態轉換與細胞移動及波型蛋白（Vimentin）表現。進一步形成腫瘤微球體後，發現抑制CCR3的表現則降低訊息傳遞分子Smad2及Akt的磷酸化受到抑制，以及癌幹細胞相關基因（Oct-4, Sox-2, Nanog）表現量皆會下降，並且微球體形成能力（sphere-forming ability）也受到抑制。此外在細胞中大量表現CCL13或以其條件培養基（Conditioned medium）做為化學吸引物（Chemoattractant）皆可促進細胞移動，並且發現大量表現CCL13會使微球體中部分幹細胞相關基因表現上升，以及微球體形成能力增加。我們也發現抑制CCR3表現可明顯減緩由CCL13介導之化學趨化性（Chemotaxis），證實CCL13之功能執行確實依賴CCR3的表現。透過Survexpress生物資訊等資料庫分析臨床相關性（clinical relevance），發現CCR3及CCL13的高表現都會使病患有高風險以及低存活的預後，與TGF-b1合併分析也發現，兩者或三者的同時高表現都會使病患有較差的預後，更加確認TGF-b1/CCR3/CCL13軸線在肺癌進程及癌幹細胞中的重要性。。
　綜合以上研究結果顯示，我們發現TGF-b1的刺激會使CCR3及CCL13表現量上升，活化下游訊息傳遞路徑如Smad及Akt，進而促進肺癌細胞的生長、移動、癌幹細胞生成及相關基因（Oct-4, Sox-2, Nanog）表現，使得病患有高風險及低存活率，我們的研究結果提供未來在肺癌治療及抑制癌幹細胞生成上一個具有潛力及可行性的新標的，而其背後機制仍待進一步的研究來闡明。

The incidence and death rates of lung cancer rank first among cancers. Poor prognosis of metastasis and recurrence are regarded to be closely associated with the presence of cancer stem cells (CSCs) in tumor. CSCs exhibit self-renewal, invasiveness, metastasis, tumorigenicity, and resistance to chemotherapy. These characteristics enable tumor cells to survive drug toxicity, thus causing metastasis and recurrence. Therefore, exploring new approaches for inhibiting the generation of CSC and expression of stem-like properties are critical issues in the development of therapeutic strategies. In recent years, many studies have reported that cancer progression is closely related to the abnormal expression of chemokines and chemokine receptors in the tumor microenvironment. The roles of chemokine family in the lung cancer progression have not been completely elucidated because of the complexities of the members and their functions.
Previous studies have demonstrated that CCR3 expression was associated with a higher grade of malignancy in glioblastoma, melanoma, and renal cell carcinoma. Moreover, CCL7 and CCL11 are known to significantly promote cell proliferation, motility, and tumor growth in a CCR3-dependent manner. Clinical research showed that CCL13 levels in serum can serve as a prognostic factor of distant metastasis in colorectal cancer, and through cytokine microarray, we found that higher levels of CCL13 were expressed in spheroid cells that were cultured in conditioned medium than in the parental cell. However, the roles of CCR3 and CCL13 in lung cancer and CSCs have not been investigated completely. Our aim was to determine the role of this chemokine axis in the fields we mentioned above.
We found that CCR3 and CCL13 mRNA levels were upregulated in both TGF-b1 treated cells and spheroids, which further confirmed their crucial role in lung cancer and CSC development. On the basis of our findings, we established stable clones of CCR3 knockdown and CCL13 overexpression through lentivirus system to investigate their biological functions. We discovered that CCR3 knockdown considerably inhibited cell growth and motility. Notably, CCR3 knockdown also attenuated TGF-b1-induced EMT morphological change and migration. Spheroid cell culture analysis revealed that the phosphorylation of signaling transducers, Smad2 and Akt, was inhibited and the expression of stemness genes, namely Oct-4, Sox-2, and Nanog, was also downregulated in CCR3 knockdown group, and the spheroid formation ability was inhibited correspondingly. CCL13 promoted cell migration either by directly overexpressing in cancer cells or as a chemoattractant secreted in the conditioned medium. Spheroid cell culture analysis revealed that CCL13 upregulated part of stemness genes and promoted spheroid formation ability. We also discovered that CCR3 knockdown significantly attenuated CCL13-induced chemotaxis, proving that CCL13 promoted cell migration in a CCR3-dependent manner. To understand the clinical relevance of CCR3 and CCL13 expression in patients with lung cancer, we analyzed the correlation of TGF-b1, CCR3, and CCL13 with overall survival of patients by using the SurvExpress database. We discovered that the expression levels were positively correlated with patient risk and negatively correlated with patient survival.
In conclusion, the stimulation of TGF-b1 in the tumor microenvironment upregulated CCR3 and CCL13 expression; activated downstream signaling transduction, such as the Smad and Akt pathway; and promoted cell growth, migration, spheroid formation, and stemness gene expression. Finally, it caused high risk and low survival in patients with lung cancer. Our research provides a potential and feasible target for treatment of lung cancer and CSC. Additional studies are required for elucidation of detailed mechanisms.

中文摘要 i
Abstract iii
目錄 v
第一章、緒論 8
1. 肺癌 (Lung cancer) 8
2. 癌幹細胞 (Cancer stem cells, CSCs) 12
3. 上皮間質轉化現象（Epithelial-mesenchymal transition, EMT） 15
4. 腫瘤微環境 (Tumor microenvironment, TME) 17
5. CCR3 20
6. CCL13 (monocyte chemoattractant protein-4, MCP-4) 24
7. 研究動機 26
第二章、材料與方法 27
第三章、實驗結果 56
第四章、討論 65
第五章、參考文獻 69
第六章、結果圖表 77
Figure 1. The expression of chemokine receptors in TGF-b1 treatment and cancer stem cells. 78
Figure 2. CCR3 knockdown efficiency in A549 and H1299 79
Figure 3. CCR3 knockdown inhibited cell proliferation. 81
Figure 4. CCR3 knockdown inhibited cell motility. 82
Figure 5. CCR3 knockdown repressed TGF-b1-promoted EMT and cell migration 85
Figure 6. CCR3 knockdown inhibited spheroid formation in TGF-b1 stimulation and stemness gene expression 87
Figure 7. CCLs expression profile in the context of A549 and H1299 stemness 88
Figure 9. CCL13 overexpression promoted spheroid formation and stemness gene expression. 93
Figure 10. CCR3 knockdown repressed CCL13-induced chemotaxis. 94
Figure 11. Higher expression of TGF-b1, CCR3 and CCL13 presented a worse survival rate in lung cancer patients. 95
Figure 12. Schematic representation of the role of CCL13/CCR3 in lung cancer progression and stemness properties. 96
第七章、附錄 97
附錄一、H1299細胞激素微陣列 97
附錄二、 RT-qPCR 各結果之 CT 值 98
附錄三、TGF-1刺激下對CCR3表現量的影響 106
附錄四、CCR3 knockdown對TGF-b1誘發之EMT細胞型態改變之影響 106
附錄五、A549 CCL13 overexpression後CCL13及CCR3蛋白表現量變化 107
附錄六、G418篩選濃度測量 107
附錄七、CCL13 overexpression對細胞生長能力的影響 108
附錄八、CCL13 overexpression對細胞移動能力的影響 109
附錄九、CCR3 表現量和病患預後之相關性分析 110
附錄十、CCL13表現量和病患預後之相關性分析 110
附錄十一、CCR3在肺癌病患組織中的表現量分析 111
附錄十二、CCR3 knockdown對細胞內ATP含量之影響 111
附錄十三、CCR3-ORF construct 定序比對結果 112
附錄十四、CCL13-ORF construct 定序比對結果 113
附表、pLAS2w.Pneo map 114
附表、pLAS2w.Pneo-CCL13 map 115
附表、pLAS2w.Ppuro map 116
附表、pLAS2w.Ppuro-CCR3 map 117
附表、肺癌細胞列表 118
附表、肺癌組織微陣列試染片樣本資料 119

[1] Siegel R, Miller K, Jemal, A. Cancer Statistics, 2018. CA Cancer J Clin 2018; 68: 7-30.
[2] Mao Y, Yang D, He J, Krasna MJ. Epidemiology of Lung Cancer. Surg Oncol Clin N Am 2016; 25: 439-45
[3] Tsao AS, Scagliotti GV, Bunn PA Jr. Scientific Advances in Lung Cancer 2015.
J Thorac Oncol 2016; 11: 613-638
[4] Hirsch FR, Scagliotti GV, Mulshine JL. Lung cancer: current therapies and new targeted treatments. Lancet 2017; 389: 299-311.
[5] Motadi LR, Misso NL, Dlamini Z, Bhoola KD. Molecular genetics and mechanisms of apoptosis in carcinomas of the lung and pleura: therapeutic targets. Int Immunopharmacol 2007; 7: 1934-47
[6] Blandin Knight S, Crosbie PA, Balata H, Chudziak J, Hussell T, Dive C. Progress and prospects of early detection in lung cancer. Open Biol. 2017; 7:
[7] Goldstraw P, Chansky K, Crowley J, Rami-Porta R. The IASLC lung cancer staging project: proposals for revision of the TNM stage groupings in the forthcoming (eighth) edition of the TNM classification for lung cancer.
J Thorac Oncol 2016; 11: 39-51
[8] Rosell R, Karachaliou N. Lung cancer in 2014: optimizing lung cancer treatment approaches. Nat Rev Clin Oncol. 2015; 12: 75-6
[9] Thomas A, Liu SV, Subramaniam DS, Giaccone G. Refining the treatment of NSCLC according to histological and molecular subtypes. Nat Rev Clin Oncol. 2015; 12: 511-26
[10] Aupérin A, Le Péchoux C, Rolland E, et al. Meta-analysis of concomitant versus sequential radiochemotherapy in locally advanced non-small-cell lung cancer. J Clin Oncol. 2010; 28: 2181-90
[11] Senan S, Brade A, Wang LH, et al. PROCLAIM: Randomized phase III trial of pemetrexed-cisplatin or etoposide-cisplatin plus thoracic radiation therapy followed by consolidation chemotherapy in locally advanced nonsquamous non-small-cell lung cancer. J Clin Oncol. 2016; 34: 953-62
[12] Zago G, Muller M, van den Heuvel M, Baas P. New targeted treatments for non-small-cell lung cancer - role of nivolumab. Biologics. 2016; 10: 103-117.
[13] Jacobsen K, Bertran-Alamillo J, Molina MA, et al. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer. Nat Commun. 2017; 8: 410
[14] Soria JC, Mauguen A, Reck M, et al. Systematic review and meta-analysis of randomised, phase II/III trials adding bevacizumab to platinum-based chemotherapy as first-line treatment in patients with advanced non-small-cell lung cancer. Ann Oncol. 2013; 24: 20-30
[15] Shaw AT, Ou SH, Bang YJ, et al. Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J Med. 2014; 371: 1963-71
[16] Kim DW, Mehra R, Tan DSW, et al. Activity and safety of ceritinib in patients with ALK-rearranged non-small-cell lung cancer (ASCEND-1): updated results from the multicentre, open-label, phase 1 trial. Lancet Oncol. 2016; 17: 452-463
[17] Ou SH, Ahn JS, De Petris L, et al. Alectinib in crizotinib-refractory ALK-rearranged non-small-cell lung cancer: A phase II global study. J Clin Oncol. 2016; 34: 661-8
[18] Heppner GH, Miller BE. Tumor heterogeneity: biological implications and therapeutic consequences. Cancer Metastasis Rev. 1983; 2 :5-23
[19] Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994; 367: 645-8
[20] Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 2008; 8: 755-68
[21] Das S, Srikanth M, Kessler JA. Cancer stem cells and glioma. Nat Clin Pract Neurol. 2008; 4: 427-35
[22] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001; 414: 105-11
[23] Nowell PC. The clonal evolution of tumor cell populations. Science. 1976; 194: 23-8
[24] Gwak JM, Kim M, Kim HJ, et al. Expression of embryonal stem cell transcription factors in breast cancer: Oct4 as an indicator for poor clinical outcome and tamoxifen resistance. Oncotarget. 2017; 8: 36305–36318.
[25] Leon G, MacDonagh L, Finn SP, Cuffe S, Barr MP. Cancer stem cells in drug resistant lung cancer: Targeting cell surface markers and signaling pathways. Pharmacol Ther. 2016; 158:71-90
[26] Begicevic RR, Falasca M. ABC transporters in cancer stem cells: beyond chemoresistance. Int J Mol Sci. 2017;18:e2362.
[27] Ho MM, Ng AV, Lam S, Hung JY. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res. 2007; 67: 4827-33
[28] Chen K, Huang YH, Chen JL. Understanding and targeting cancer stem cells: therapeutic implications and challenges. Acta Pharmacol Sin. 2013; 34: 732-40
[29] Ishiguro T, Ohata H, Sato A, Yamawaki K, Enomoto T, Okamoto K. Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications. Cancer Sci. 2017; 108: 283-289
[30] Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994; 367: 645-8
[31] Clevers H. The cancer stem cell: premises, promises and challenges. Nat Med. 2011; 17: 313-9
[32] Singh SK, Clarke ID, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003; 63: 5821-8
[33] Pastrana E, Silva-Vargas V, Doetsch F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell. 2011; 8: 486-98
[34] Clarke MF, Dick JE, Dirks PB, et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006; 66: 9339-44
[35] Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2014; 15: 178-96
[36] Xu J, Lamouille S, Derynck R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res. 2009; 19: 156-72
[37] Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol. 2006; 7: 131-42
[38] Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development. 2005; 132: 3151-61
[39] Cano A, Pérez-Moreno MA, Rodrigo I, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000; 2: 76-83
[40] Yang F, Sun L, Li Q, et al. SET8 promotes epithelial-mesenchymal transition and confers TWIST dual transcriptional activities. EMBO J. 2012; 31: 110-23
[41] Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133(4):704-15
[42] Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer. 2009; 9: 239-52
[43] Derynck R, Goeddel DV, Ullrich A, et al. Synthesis of messenger RNAs for transforming growth factors alpha and beta and the epidermal growth factor receptor by human tumors. Cancer Res. 1987;47(3):707-12
[44] Dickson RB, Kasid A, Huff KK, et al. Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17 beta-estradiol or v-Ha-ras oncogene. Proc Natl Acad Sci U S A. 1987;84(3):837-41
[45] Datto MB, Li Y, Panus JF, Howe DJ, Xiong Y, Wang XF. Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism. Proc Natl Acad Sci U S A. 1995; 92: 5545-9
[46] Papageorgis P. TGFβ Signaling in Tumor Initiation, Epithelial-to-Mesenchymal Transition, and Metastasis. J Oncol. 2015;2015 587193.
[47] Zhang YE. Non-Smad pathways in TGF-beta signaling. Cell Res. 2009; 19: 128-39
[48] Siegel PM, Massagué J. Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer. 2003; 3: 807-21
[49] Zlotnik A, Burkhardt AM, Homey B. Homeostatic chemokine receptors and organ-specific metastasis. Nat Rev Immunol. 2011; 11: 597-606
[50] Nagarsheth N, Wicha MS, Zou W. Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy. Nat Rev Immunol. 2017; 17: 559-572
[51] de Munnik SM, Smit MJ, Leurs R, Vischer HF. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors. Front Pharmacol. 2015; 6:40
[52] Wise A, Gearing K, Rees S. Target validation of G-protein coupled receptors. Drug Discov Today. 2002; 7: 235-46
[53] Zimmermann N, Colyer JL, Koch LE, Rothenberg ME. Analysis of the CCR3 promoter reveals a regulatory region in exon 1 that binds GATA-1. BMC Immunol. 2005;6:7.
[54] Joubert P, Lajoie-Kadoch S, Labonté I, et al. CCR3 expression and function in asthmatic airway smooth muscle cells. J Immunol. 2005; 175: 2702-8
[55] Uguccioni M, Mackay CR, Ochensberger B, et al. High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines. J Clin Invest. 1997; 100: 1137-43
[56] Kampen GT, Stafford S, Adachi T, et al. Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases. Blood. 2000; 95: 1911-7
[57] Fujisawa T, Kato Y, Nagase H, et al. Chemokines induce eosinophil degranulation through CCR-3. J Allergy Clin Immunol. 2000; 106: 507-13
[58] Erin EM, Williams TJ, Barnes PJ, Hansel TT. Eotaxin receptor (CCR3) antagonism in asthma and allergic disease. Curr Drug Targets Inflamm Allergy. 2002; 1: 201-14
[59] Gaspar K, Kukova G, Bunemann E, et al. The chemokine receptor CCR3 participates in tissue remodeling during atopic skin inflammation. J Dermatol Sci. 2013; 71: 12-21
[60] Agrawal L, Maxwell CR, Peters PJ, et al. Complexity in human immunodeficiency virus type 1 (HIV-1) co-receptor usage: roles of CCR3 and CCR5 in HIV-1 infection of monocyte-derived macrophages and brain microglia. J Gen Virol. 2009; 90: 710-22
[61] Jöhrer K, Zelle-Rieser C, Perathoner A, et al. Up-regulation of functional chemokine receptor CCR3 in human renal cell carcinoma. Clin Cancer Res. 2005; 11: 2459-65
[62] Lee YJ, Kim DH, Lee SH, Nam HS, Roh MR, Cho MK. Chemokine Receptor CCR3 Expression in Malignant Cutaneous Tumors. Ann Dermatol. 2010; 22: 412-7
[63] Kouno J, Nagai H, Nagahata T, et al. Up-regulation of CC chemokine, CCL3L1, and receptors, CCR3, CCR5 in human glioblastoma that promotes cell growth. J Neurooncol. 2004; 70: 301-7
[64] Tian M, Chen L, Ma L, et al. Expression and prognostic significance of CCL11/CCR3 in glioblastoma. Oncotarget. 2016; 7: 32617-27
[65] Laurent V, Guérard A, Mazerolles C, et al. Periprostatic adipocytes act as a driving force for prostate cancer progression in obesity. Nat Commun. 2016
[66] Lee YS, Kim SY, Song SJ, et al. Crosstalk between CCL7 and CCR3 promotes metastasis of colon cancer cells via ERK-JNK signaling pathways. Oncotarget. 2016; 7: 36842-36853
[67] Zhu F, Liu P, Li J, Zhang Y. Eotaxin-1 promotes prostate cancer cell invasion via activation of the CCR3-ERK pathway and upregulation of MMP-3 expression. Oncol Rep. 2014; 31: 2049-54
[68] Miyagaki T, Sugaya M, Murakami T, et al. CCL11-CCR3 interactions promote survival of anaplastic large cell lymphoma cells via ERK1/2 activation. Cancer Res. 2011; 71: 2056-65
[69] Salcedo R, Young HA, Ponce ML, et al. Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells. J Immunol. 2001; 166: 7571-8
[70] Huang G, Tao L, Shen S, Chen L. Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells. Sci Rep. 2016; 6: 27152.
[71] Pease JE, Horuk R. Recent progress in the development of antagonists to the chemokine receptors CCR3 and CCR4. Expert Opin Drug Discov. 2014; 9: 467-83
[72] Lamkhioued B, Garcia-Zepeda EA, Abi-Younes S, et al. Monocyte chemoattractant protein (MCP)-4 expression in the airways of patients with asthma. Induction in epithelial cells and mononuclear cells by proinflammatory cytokines. Am J Respir Crit Care Med. 2000; 162: 723-32
[73] Hein H, Schlüter C, Kulke R, Christophers E, Schröder JM, Bartels J. Genomic organization, sequence analysis and transcriptional regulation of the human MCP-4 chemokine gene (SCYA13) in dermal fibroblasts: a comparison to other eosinophilic beta-chemokines. Biochem Biophys Res Commun. 1999; 255: 470-6
[74] Garcia-Zepeda EA, Combadiere C, Rothenberg ME, et al. Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3. J Immunol. 1996; 157: 5613-26
[75] Panelli MC, White R, Foster M, et al. Forecasting the cytokine storm following systemic interleukin (IL)-2 administration. J Transl Med. 2004; 2: 17
[76] Lambrecht BN, Hammad H. The airway epithelium in asthma. Nat Med. 2012; 18: 684-92
[77] Tenscher K, Metzner B, Hofmann C, Schöpf E, Norgauer J. The monocyte chemotactic protein-4 induces oxygen radical production, actin reorganization, and CD11b up-regulation via a pertussis toxin-sensitive G-protein in human eosinophils. Biochem Biophys Res Commun. 1997; 240: 32-5
[78] Kohan M, Puxeddu I, Reich R, Levi-Schaffer F, Berkman N. Eotaxin-2/CCL24 and eotaxin-3/CCL26 exert differential profibrogenic effects on human lung fibroblasts. Ann Allergy Asthma Immunol. 2010; 104: 66-72
[79] Dean RA, Cox JH, Bellac CL, Doucet A, Starr AE, Overall CM. Macrophage-specific metalloelastase (MMP-12) truncates and inactivates ELR+ CXC chemokines and generates CCL2, -7, -8, and -13 antagonists: potential role of the macrophage in terminating polymorphonuclear leukocyte influx. Blood. 2008; 112: 3455-64
[80] Martínez-Becerra F, Silva DA, Domínguez-Ramírez L, et al. Analysis of the antimicrobial activities of a chemokine-derived peptide (CDAP-4) on Pseudomonas aeruginosa. Biochem Biophys Res Commun. 2007; 355: 352-8
[81] Iwamoto T, Okamoto H, Kobayashi S, et al. A role of monocyte chemoattractant protein-4 (MCP-4)/CCL13 from chondrocytes in rheumatoid arthritis. FEBS J. 2007; 274: 4904-12
[82] Yamaguchi A, Nozawa K, Fujishiro M, et al. CC motif chemokine ligand 13 is associated with rheumatoid arthritis pathogenesis. Mod Rheumatol. 2013; 23: 856-63
[83] Inoue Y, Tsushima H, Ando K, et al. Chemokine expression in human erythroid leukemia cell line AS-E2: macrophage inflammatory protein-3alpha/CCL20 is induced by inflammatory cytokines. Exp Hematol. 2006; 34: 19-26
[84] Chen Q, Zheng T, Lan Q, et al. Single-nucleotide polymorphisms in genes encoding for CC chemokines were not associated with the risk of non-Hodgkin lymphoma. Cancer Epidemiol Biomarkers Prev. 2013; 22: 1332-5
[85] Okugawa Y, Toiyama Y, Mohri Y, et al. Elevated serum concentration of monocyte chemotactic protein 4 (MCP-4) as a novel non-invasive prognostic and predictive biomarker for detection of metastasis in colorectal cancer. J Surg Oncol. 2016; 114: 483-9
[86] Hou YL, Chen H, Dong ZH, et al. Clinical significance of serum transforming growth factor-β1 in lung cancer. Cancer Epidemiol. 2013; 37: 750-3
[87] Zhu XH, Liao B, Xu Y, et al. Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils. Mol Med Rep. 2017; 15: 696-702
[88] Sato K, Kawasaki H, Nagayama H, et al. TGF-beta 1 reciprocally controls chemotaxis of human peripheral blood monocyte-derived dendritic cells via chemokine receptors. J Immunol. 2000; 164: 2285-95
[89] Kitayama J, Mackay CR, Ponath PD, Springer TA. The C-C chemokine receptor CCR3 participates in stimulation of eosinophil arrest on inflammatory endothelium in shear flow. J Clin Invest. 1998; 101: 2017-24