臺灣博碩士論文加值系統

大腸直腸腫瘤細胞的轉移成功與否受液體機械力所調控，而大腸直腸癌的高轉移率為大腸直腸癌患者的死亡主因。因此，本研究將探討流體壓力中的液體機械力對大腸直腸癌轉移相關惡性能力之影響。大腸直腸腫瘤SW620細胞在蠕動幫浦所供應的液體機械力分別處理5分鐘、1小時以及2小時，其不變之存活率、低壞死與凋亡細胞率，及較高的peNOSSer1177蛋白質表現量，顯示流體裝置在處理細胞上的可靠度。在分析粒線體功能上，發現細胞ATP總量不變，但粒線體膜電位上升。細胞凋亡蛋白質，caspase 9，表現量也上升。細胞型態上，細胞碎片量與表面複雜度的提升顯示細胞受損。上皮-間質轉換蛋白質， E-CAD和ZO-1，的上升，以及細胞週期在S期的停滯，都顯示了液體機械力降低大腸直腸癌之惡性度。為進一步確認液體機械力對SW620惡性度的調控，在SW620經液體機械力處理終止後48小時，分析發現粒線體膜電位下降、細胞caspase 9表現量不變、E-CAD和ZO-1表現量降低、細胞週期恢復進行，且細胞侵襲能力和腫瘤團簇形成能力在停止液體機械力後上升。以上與流體壓力處理下相反之結果與流體壓力終止後惡性度的上升，均顯示了流體壓力為調控大腸直腸腫瘤細胞惡性度的關鍵。然而，胃腫瘤細胞之EMT在相同流體壓力處理下被促進，eNOS蛋白質也並無顯著改變趨勢。總結，液體機械力降低了大腸直腸癌的惡性度，但促進了胃癌惡性度。本研究在未來也想進一步確認液體機械力所調控之機械受器蛋白為何及其所帶來之影響。

Blood flow-derived mechanical forces are key factors for the successful metastasis of CRC. And high metastatic potential is the main cause of colorectal cancer (CRC) mortality. Therefore, the study proposed the fluid flow effects on the physiological responses of CRC cells. CRC cell SW620 subjected to fluid flow within 2 hours generated by a rotation pump show a high survival rate, low necrosis ratio, and low apoptosis ratio. Following an elevation of peNOSSer1177 and regulation of nitric oxide (NO) that shows the sensing of fluid-derived mechanical stimuli in SW620 cells, indicated the suitability of the flow device. Further experiments showed that the levels of ATP remained constant, as the mitochondrial membrane potential (MMP) burst under flow treatment. An increase of apoptosis initiator, caspase 9, and the increase of cell surface complexity and cell fragment, indicates an injury of CRC. All above, accompanied by the upregulation of epithelial phenotype markers, E-cadherin (E-CAD) and Zonula occludens-1 (ZO-1), showing downregulation of epithelial-mesenchymal transition (EMT), and the cell cycle arrest in the S phase demonstrate the downregulation of CRC malignancy under flow treatment. To confirm the effect of fluid flow on the malignance of SW620. To further confirm the effect of flow-derived mechanical forces on the tumor cell, SW620 was cultured under a static state after 0, 5, 60, and 120 mins of fluid flow treatment. The expression of eNOS and caspase 9 between different groups show no difference, as the nitric oxide production rate, MMP change, and expression of E-CAD and ZO-1 protein were found in an opposite tendency compared to the tendency under fluid flow treatment. Whereas the invasion of SW620 was enhanced. The spheroid-forming cell number of the tumor was induced after the termination of fluid flow. Indicating the importance of fluid mechanical force on the malignancy of colorectal cancer. To conclude, flow-derived mechanical forces downregulated the malignancy of CRC. However, induction of EMT was found in gastric cancer under the same flow treatment, indicating an increase in the malignancy of gastric cancer. Showing the organ-specific effect of fluid flow treatment on cancer. Therefore, we will further investigate the specific mechanical receptors that were modulated under fluid flow treatment.

論文通過合格證書………………………………………………………………….…..I
無違反學術倫理聲明書……………………………………………………………….II
中文摘要……………………………………………………….…………….……….. III
英文摘要……………………………………………………………….…...….……...IV
致謝辭…………………………………………………………………...……..............V
目次………………………………………………………………….….….................VII
縮寫表………………………………………………………………………………....IX
前言…………………………………………………………………….…..................XI
第一章、 緒論…………………………………………………………....................1
第一節、 尋找抑制大腸直腸癌轉移治療方法之急迫性……….....................1
第二節、 循環腫瘤細胞之研究成果…………………………….....................2
第三節、 腫瘤細胞調節機械力之機制………………………….....................4
第四節、 eNOS蛋白質及一氧化氮表現量與機械訊號偵測之關聯性...........6
第五節、 腫瘤細胞不同惡性能力被不同條件之流體壓力所促進..................9
第六節、 以細胞株觀察不同流體壓力對腫瘤細胞惡性能力之影響….........11
第二章、 研究目的………………………………………………….…...................12
第三章、 研究材料與方法…………………………………………........................13
第四章、 研究結果…………………………………………………........................32
第五章、 結論………………………………………………………........................39
第六章、 討論………………………………………………………........................39
第一節、 流體壓力下之機械訊號受器與未來期許………………………….39
第二節、 血壓與罹癌率之關聯性……………………………….....................39
第三節、 CyclinB1與CyclinD1的表現和細胞週期停滯之關聯性...............40
第四節、 流體壓力下細胞自噬的發生……………………………………….41
第五節、 上皮間質型態轉換的可塑性…………...…………..........................42
第六節、 腫瘤團簇形成能力在經流體壓力處理過之細胞較高的可能原因.43
第七節、 eNOS蛋白磷酸化程度與一氧化氮表現量趨勢不同之原因探討..44
第八節、 粒線體膜電位受流體壓力調控下與ATP改變趨勢的不同………45
第九節、 細胞表面複雜度與碎片形成比例之變化對細胞生存率之影響.....46
第十節、 流體壓力對腫瘤細胞影響之器官、組織特異性…………...……..48
實驗結果圖………………………………………………………................................50
第一節、 流體壓力處理裝置之可靠度…….……………….….......................50
第二節、 流體壓力降低大腸直腸腫瘤細胞之惡性度...………….................54
第三節、 流體壓力影響腫瘤細胞惡性度之重要性驗證...……….................59
第四節、 流體壓力對腫瘤惡性度影響之器官特異性………………………71
參考文獻…………………………………………………………...............................80

1.Dekker, E., et al., Colorectal cancer. Lancet, 2019. 394(10207): p. 1467-1480.
2.Hu, Z., et al., Quantitative evidence for early metastatic seeding in colorectal cancer. Nat Genet, 2019. 51(7): p. 1113-1122.
3.Desmeules, P., et al., Retrospective Assessment of Complementary Liquid Biopsy on Tissue Single-Gene Testing for Tumor Genotyping in Advanced NSCLC. Curr Oncol, 2023. 30(1): p. 575-585.
4.Arnold, C. and P. Webster, 11 clinical trials that will shape medicine in 2023. Nat Med, 2022. 28(12): p. 2444-2448.
5.Blumen, H., K. Fitch, and V. Polkus, Comparison of Treatment Costs for Breast Cancer, by Tumor Stage and Type of Service. Am Health Drug Benefits, 2016. 9(1): p. 23-32.
6.Mantovani, F., L. Collavin, and G. Del Sal, Mutant p53 as a guardian of the cancer cell. Cell Death Differ, 2019. 26(2): p. 199-212.
7.Follain, G., et al., Fluids and their mechanics in tumour transit: shaping metastasis. Nat Rev Cancer, 2020. 20(2): p. 107-124.
8.Lin, D., et al., Circulating tumor cells: biology and clinical significance. Signal Transduct Target Ther, 2021. 6(1): p. 404.
9.Janni, W.J., et al., Pooled Analysis of the Prognostic Relevance of Circulating Tumor Cells in Primary Breast Cancer. Clin Cancer Res, 2016. 22(10): p. 2583-93.
10.Anvari, S., E. Osei, and N. Maftoon, Interactions of platelets with circulating tumor cells contribute to cancer metastasis. Sci Rep, 2021. 11(1): p. 15477.
11.Gkountela, S., et al., Circulating Tumor Cell Clustering Shapes DNA Methylation to Enable Metastasis Seeding. Cell, 2019. 176(1-2): p. 98-112.e14.
12.Aceto, N., Bring along your friends: Homotypic and heterotypic circulating tumor cell clustering to accelerate metastasis. Biomed J, 2020. 43(1): p. 18-23.
13.Qazi, H., Z.D. Shi, and J.M. Tarbell, Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression. PLoS One, 2011. 6(5): p. e20348.
14.Yankaskas, C.L., et al., The fluid shear stress sensor TRPM7 regulates tumor cell intravasation. Sci Adv, 2021. 7(28).
15.Ring, A., et al., Biology, vulnerabilities and clinical applications of circulating tumour cells. Nat Rev Cancer, 2023. 23(2): p. 95-111.
16.Angulo-Urarte, A., T. van der Wal, and S. Huveneers, Cell-cell junctions as sensors and transducers of mechanical forces. Biochim Biophys Acta Biomembr, 2020. 1862(9): p. 183316.
17.Fares, J., et al., Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther, 2020. 5(1): p. 28.
18.Po, J.W., et al., Improved ovarian cancer EMT-CTC isolation by immunomagnetic targeting of epithelial EpCAM and mesenchymal N-cadherin. J Circ Biomark, 2018. 7: p. 1849454418782617.
19.Negishi, R., et al., Transcriptomic profiling of single circulating tumor cells provides insight into human metastatic gastric cancer. Commun Biol, 2022. 5(1): p. 20.
20.Alvarado-Estrada, K., et al., Circulatory shear stress induces molecular changes and side population enrichment in primary tumor-derived lung cancer cells with higher metastatic potential. Sci Rep, 2021. 11(1): p. 2800.
21.Brabletz, S., et al., Dynamic EMT: a multi-tool for tumor progression. Embo j, 2021. 40(18): p. e108647.
22.Hope, J.M., et al., Fluid shear stress enhances T cell activation through Piezo1. BMC Biol, 2022. 20(1): p. 61.
23.Xu, S., et al., New insights into shear stress-induced endothelial signalling and barrier function: cell-free fluid versus blood flow. Cardiovasc Res, 2017. 113(5): p. 508-518.
24.Kuck, L., et al., Shear Stress Ameliorates Superoxide Impairment to Erythrocyte Deformability With Concurrent Nitric Oxide Synthase Activation. Front Physiol, 2019. 10: p. 36.
25.Nagarajan, S., et al., Mechanical perturbations trigger endothelial nitric oxide synthase activity in human red blood cells. Sci Rep, 2016. 6: p. 26935.
26.Fleming, I., Molecular mechanisms underlying the activation of eNOS. Pflugers Arch, 2010. 459(6): p. 793-806.
27.Gayer, C.P. and M.D. Basson, The effects of mechanical forces on intestinal physiology and pathology. Cell Signal, 2009. 21(8): p. 1237-44.
28.Follain, G., et al., Hemodynamic Forces Tune the Arrest, Adhesion, and Extravasation of Circulating Tumor Cells. Dev Cell, 2018. 45(1): p. 33-52.e12.
29.Zou, D., et al., Pan-Cancer Analysis of NOS3 Identifies Its Expression and Clinical Relevance in Gastric Cancer. Front Oncol, 2021. 11: p. 592761.
30.Lim, K.H., et al., Tumour maintenance is mediated by eNOS. Nature, 2008. 452(7187): p. 646-9.
31.Kashfi, K., Nitric oxide in cancer and beyond. Biochem Pharmacol, 2020. 176: p. 114006.
32.Stoner, L., et al., The importance of velocity acceleration to flow-mediated dilation. Int J Vasc Med, 2012. 2012: p. 589213.
33.Huang, Q., et al., Shear stress activates ATOH8 via autocrine VEGF promoting glycolysis dependent-survival of colorectal cancer cells in the circulation. J Exp Clin Cancer Res, 2020. 39(1): p. 25.
34.Shiue, T.W., et al., Nitric oxide turn-on fluorescent probe based on deamination of aromatic primary monoamines. Inorg Chem, 2012. 51(9): p. 5400-8.
35.Chen, P.H., et al., Hydrogen sulfide increases nitric oxide production and subsequent S-nitrosylation in endothelial cells. ScientificWorldJournal, 2014. 2014: p. 480387.
36.Weth, A., et al., A novel device for elimination of cancer cells from blood specimens. Sci Rep, 2020. 10(1): p. 10181.
37.Cornelison, R.C., et al., Convective forces increase CXCR4-dependent glioblastoma cell invasion in GL261 murine model. Sci Rep, 2018. 8(1): p. 17057.
38.Fan, R., et al., Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells. Sci Rep, 2016. 6: p. 27073.
39.Gala de Pablo, J., et al., Biochemical fingerprint of colorectal cancer cell lines using label-free live single-cell Raman spectroscopy. J Raman Spectrosc, 2018. 49(8): p. 1323-1332.
40.Roper, S.J., et al., 3D spheroid models of paediatric SHH medulloblastoma mimic tumour biology, drug response and metastatic dissemination. Sci Rep, 2021. 11(1): p. 4259.
41.Kuburich, N.A., et al., In Vitro Quantification of Cancer Stem Cells Using a Mammosphere Formation Assay. Methods Mol Biol, 2022. 2429: p. 509-513.
42.Zhao, J., et al., Separation and single-cell analysis for free gastric cancer cells in ascites and peritoneal lavages based on microfluidic chips. EBioMedicine, 2023. 90: p. 104522.
43.Antunes, T.T., et al., Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension. Hypertension, 2016. 67(4): p. 763-73.
44.Seretis, A., et al., Association between blood pressure and risk of cancer development: a systematic review and meta-analysis of observational studies. Sci Rep, 2019. 9(1): p. 8565.
45.Connaughton, M. and M. Dabagh, Association of Hypertension and Organ-Specific Cancer: A Meta-Analysis. Healthcare (Basel), 2022. 10(6).
46.Cohen, J.B., et al., Cancer Therapy-Related Hypertension: A Scientific Statement From the American Heart Association. Hypertension, 2023. 80(3): p. e46-e57.
47.Małyszko, J., et al., Hypertension in malignancy-an underappreciated problem. Oncotarget, 2018. 9(29): p. 20855-20871.
48.Österlund, P., et al., Hypertension and overall survival in metastatic colorectal cancer patients treated with bevacizumab-containing chemotherapy. Br J Cancer, 2011. 104(4): p. 599-604.
49.Chang, S.F., et al., Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad. Proc Natl Acad Sci U S A, 2008. 105(10): p. 3927-32.
50.Gavet, O. and J. Pines, Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis. J Cell Biol, 2010. 189(2): p. 247-59.
51.Jin, P., S. Hardy, and D.O. Morgan, Nuclear localization of cyclin B1 controls mitotic entry after DNA damage. J Cell Biol, 1998. 141(4): p. 875-85.
52.Chen, K., et al., The membrane-associated form of cyclin D1 enhances cellular invasion. Oncogenesis, 2020. 9(9): p. 83.
53.Lee, Y.H., C.W. Lai, and Y.C. Cheng, Fluid Shear Stress Induces Cell Cycle Arrest in Human Urinary Bladder Transitional Cell Carcinoma Through Bone Morphogenetic Protein Receptor-Smad1/5 Pathway. Cell Mol Bioeng, 2018. 11(3): p. 185-195.
54.Qin, X., et al., MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells. Proc Natl Acad Sci U S A, 2010. 107(7): p. 3240-4.
55.Saraste, A., Morphologic criteria and detection of apoptosis. Herz, 1999. 24(3): p. 189-95.
56.Regmi, S., A. Fu, and K.Q. Luo, High Shear Stresses under Exercise Condition Destroy Circulating Tumor Cells in a Microfluidic System. Sci Rep, 2017. 7: p. 39975.
57.Feng, Y., et al., Timing of apoptosis onset depends on cell cycle progression in peripheral blood lymphocytes and lymphocytic leukemia cells. Oncol Rep, 2007. 17(6): p. 1437-44.
58.Mahmoud, M.M., et al., Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail. Sci Rep, 2017. 7(1): p. 3375.
59.Rajendran, V. and M.V. Jain, In Vitro Tumorigenic Assay: Colony Forming Assay for Cancer Stem Cells. Methods Mol Biol, 2018. 1692: p. 89-95.
60.Liu, X., et al., Epithelial-type systemic breast carcinoma cells with a restricted mesenchymal transition are a major source of metastasis. Sci Adv, 2019. 5(6): p. eaav4275.
61.Chen, B.J., et al., What makes leader cells arise: Intrinsic properties and support from neighboring cells. J Cell Physiol, 2020. 235(12): p. 8983-8995.
62.Vilchez Mercedes, S.A., et al., Decoding leader cells in collective cancer invasion. Nat Rev Cancer, 2021. 21(9): p. 592-604.
63.Barbazán, J., et al., Molecular characterization of circulating tumor cells in human metastatic colorectal cancer. PLoS One, 2012. 7(7): p. e40476.
64.Cuvelier, M., et al., Distribution and propagation of mechanical stress in simulated structurally heterogeneous tissue spheroids. Soft Matter, 2021. 17(27): p. 6603-6615.
65.Basehore, S.E., et al., Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity. Circ Res, 2021. 129(11): p. 1054-1066.
66.Aulak, K.S., et al., Specific O-GlcNAc modification at Ser-615 modulates eNOS function. Redox Biol, 2020. 36: p. 101625.
67.Xu, W., et al., The role of nitric oxide in cancer. Cell Res, 2002. 12(5-6): p. 311-20.
68.Wang, H., et al., Nitric Oxide (NO) and NO Synthases (NOS)-Based Targeted Therapy for Colon Cancer. Cancers (Basel), 2020. 12(7).
69.Zorova, L.D., et al., Mitochondrial membrane potential. Anal Biochem, 2018. 552: p. 50-59.
70.Lammerding, J. and R.T. Lee, Torn apart: membrane rupture in muscular dystrophies and associated cardiomyopathies. J Clin Invest, 2007. 117(7): p. 1749-52.
71.Marshall, W.F., et al., What determines cell size? BMC Biol, 2012. 10: p. 101.
72.Perea Paizal, J., S.H. Au, and C. Bakal, Squeezing through the microcirculation: survival adaptations of circulating tumour cells to seed metastasis. Br J Cancer, 2021. 124(1): p. 58-65.
73.Huang, Q., et al., Fluid shear stress and tumor metastasis. Am J Cancer Res, 2018. 8(5): p. 763-777.
74.Panciera, T., et al., Mechanobiology of YAP and TAZ in physiology and disease. Nat Rev Mol Cell Biol, 2017. 18(12): p. 758-770.