臺灣博碩士論文加值系統

癌細胞的微環境，被認為是影響癌症進程的重要指標，研究顯示，由於癌細胞特殊的代謝機制，細胞胞外基質有相比於內部更高的氫離子濃度。而癌細胞暴露於這種環境下會對其功能產生影響，其影響包括了代謝系統的轉變、生長進程的介導與自噬作用蛋白的表現。而相比於身體其他部位的癌症，口腔作為消化系統對外攝食的唯一入口，相比於生長在身體其他部位，口腔內的癌細胞有更高的頻率遭受外界環境所刺激影響，尤其是因應消化食物所分泌的唾液而產生偏酸性的環境。因此口腔癌細胞不只因應內源性的細胞周微酸化，更受到外源性口腔消化系統的影響。所以對於口腔中酸性刺激之於癌細胞的改變應當作為口腔癌發展考量的其一要素，然目前卻鮮少有以口腔癌作為模型的微酸化環境相關研究。
本篇論文以微酸性環境對癌細胞生長的影響為主軸，探討長期在酸性培養基所誘導的酸化口腔癌細胞，經由一系列的體外實驗(in vitro)與將腫瘤植入裸鼠皮下的體內(in vivo)的相互應證，闡述酸性刺激是否會影響癌細胞的幹性進而提升其抵抗環境的能力。
結果顯示，酸化對於不同種類的口腔癌細胞，有著顯著的差異，對於舌癌(SAS)，長期的酸性刺激會增加其幹性能力進而提升細胞對於化療藥物的抵抗能力，且對於擬態血管的生成有較好的表現能力; 但酸性刺激對於口腔鱗狀細胞癌(OECM1)，卻有著截然不同的影響，酸性刺激後，在體外幹性功能的表現能力反而降低，且移植進裸鼠體內表現與預期相差甚遠，大部分移植部位甚至沒有腫瘤長出，這可能與其細胞無表現擬態血管進而影響早期腫瘤發展有關。

The microenvironment of cancer cells is considered to be an important indicator of cancer progression. Studies have shown that due to the specific metabolic mechanisms of cancer cells, the extracellular matrix of cell has a higher hydrogen ion concentration than the cytoplasm. Exposure of cancer cells to this environment has an effect on their function, including changing in the metabolic system, mediating the growth processes, and expression of autophagy proteins. Compared with cancer within other parts of the body, the oral cavity is the only access to the digestive system. The cancer cells that grow in are more frequently affected by the external environment, especially with acidic environment due to food digestion of secreting seliva. Therefore, Oral cancer cells are not only influenced by endogenous micro-acidification, but also by the exogenous oral digestive system, we presumed that oral cancer should have higher research value in related field about how the microenvironment acidosis changing cell performance. However, there are few related studies using oral cancer as a subject.
In this thesis, the influence of the slightly acidic environment on the growth of cancer cells is taken as the main story. We collaboration in vivo and in vitro experiment. Observing the performance of stemness and extented to drug resistance, proofing that both in vivo and in vitro test can show consistent results.
The results show that acidification has significant impact on different types of oral cancer cells line. For tongue cancer (SAS), long-term acid stimulation its ability to upregulate stemness and further have its influence on proliferation and chemoresistance, at the meanwhile acidosis cancer cell can also improve its vasculogenic mimicry ability. But the acidosis stimulation has totally different influence on oral squamous cell carcinoma (OECM1), stemness of the OEMC1 was down regulation after acid treated but proliferation and chemoresistance ability was same as SAS. Nevertheless, OECM1 in vivo tumor incident rate showed dramatic different from in vitro side population data that OECM1 had much more lower tumor incident rate than SAS. Leak of vasculogenic mimicry ability may be one of the reason that tumor couldn’t form enough vascular-like tube to gain nutrient and lead awful in vivo tumor incident rate.

致謝 II
中文摘要 III
ABSTRACT IV
CONTENTS VI
INTRODUCTION 1
1.1 TUMOR MICROENVIRONMENT(TME) 1
1.2 MICROENVIRONMENT ACIDOSIS 2
1.3 TME ACIDOSIS RESEARCH IN COMMON CANCERS 3
1.4 UNIQUE PH CHALLENGE OF THE ORAL CAVITY 6
1.5 ORAL CANCER RECURRENCE RATE 7
1.6 CANCER STEM CELL 8
1.7 HYPOTHESIS 8
MATERIALS AND METHODS 10
2.1 CELL LINE 10
2.2 CELL CULTURE 10
2.3 CELL PROLIFERATION ASSAY 11
2.4 SIDE POPULATION 11
2.5 TUBE FORMATION ASSAY 12
2.6 XENOGRAFT IMPLANTATION 12
2.7 H&E STAINING 13
2.8. IMMUNOHISTOCHEMISTRY 13
RESULT 15
3.1 IN VITRO: ACIDOSIS SAS EXPRESS HIGHLY STEMNESS BUT OECM1 HAVE REVERSELY CONSEQUENCE 15
3.2 IN VITRO: ACIDOSIS CELL POSSESS HIGHER PROLIFERATION RATE AND CHEMORESISTANCE ABILITY 16
3.3 IN VIVO: ACIDOSIS CELL SHOW SLIGHTLY MORE TUMOR INCIDENT RATE COMPARED WITH WILD TYPE. 17
3.4 IN VIVO: ACIDOSIS SAS TUMOR PERFORM HIGHER TUMOR GROWTH RATE AND LOWER DRUG SENSITIVITY 18
3.5 IN VIVO TUMOR SPECIMEN (SAS): TUMOR FROM ACIDOSIS CELL POSSESS MORE VAST SOLID REGION AND MORE VASCULAR FORMATION 19
3.6 IN VITRO: SAS ACIDOSIS CELL PERFORM HIGHER TUBE FORMATION ABILITY, BUT THERE ARE SCARCELY OBSERVED IN OECM1 CELL LINE 21
CONCLUSION 23
DISCUSSION 24
FUTURE WORK 27
REFERENCE 28
FIGURES AND LEGENDS 31
APPENDIX 44

1. Balkwill, F.R., M. Capasso, and T. Hagemann, The tumor microenvironment at a glance. J Cell Sci, 2012. 125(Pt 23): p. 5591-6.
2. Hanahan, D. and L.M. Coussens, Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell, 2012. 21(3): p. 309-22.
3. J. Bissell, M., H.G. Hall, and G. Parry, Bissell MJ, Hall HG, Parry G.. How does the extracellular matrix direct gene expression? J Theor Biol 99: 31-68. Vol. 99. 1982. 31-68.
4. Armulik, A., A. Abramsson, and C. Betsholtz, Endothelial/pericyte interactions. Circ Res, 2005. 97(6): p. 512-23.
5. Navas, C., et al., EGF receptor signaling is essential for k-ras oncogene-driven pancreatic ductal adenocarcinoma. Cancer Cell, 2012. 22(3): p. 318-30.
6. Cirri, P. and P. Chiarugi, Cancer-associated-fibroblasts and tumour cells: a diabolic liaison driving cancer progression. Cancer Metastasis Rev, 2012. 31(1-2): p. 195-208.
7. Mohamed, M.M. and B.F. Sloane, Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer, 2006. 6(10): p. 764-75.
8. Pontiggia, O., et al., The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through β1 integrin. Breast cancer research and treatment, 2012. 133(2): p. 459-471.
9. De Bock, K., S. Cauwenberghs, and P. Carmeliet, Vessel abnormalization: another hallmark of cancer? Molecular mechanisms and therapeutic implications. Curr Opin Genet Dev, 2011. 21(1): p. 73-9.
10. Goel, S., et al., Normalization of the vasculature for treatment of cancer and other diseases. Physiol Rev, 2011. 91(3): p. 1071-121.
11. Takeda, N., et al., Differential activation and antagonistic function of HIF-{alpha} isoforms in macrophages are essential for NO homeostasis. Genes Dev, 2010. 24(5): p. 491-501.
12. Pietras, K. and A. Ostman, Hallmarks of cancer: interactions with the tumor stroma. Exp Cell Res, 2010. 316(8): p. 1324-31.
13. Martinez-Outschoorn, U.E., F. Sotgia, and M.P. Lisanti, Power surge: supporting cells "fuel" cancer cell mitochondria. Cell Metab, 2012. 15(1): p. 4-5.
14. Nieman, K.M., et al., Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med, 2011. 17(11): p. 1498-503.
15. Manzur, M., J. Hamzah, and R. Ganss, Modulation of the "blood-tumor" barrier improves immunotherapy. Cell Cycle, 2008. 7(16): p. 2452-2455.
16. Sennino, B. and D.M. McDonald, Controlling escape from angiogenesis inhibitors. Nat Rev Cancer, 2012. 12(10): p. 699-709.
17. Bertout, J.A., S.A. Patel, and M.C. Simon, The impact of O2 availability on human cancer. Nat Rev Cancer, 2008. 8(12): p. 967-75.
18. Corbet, C. and O. Feron, Tumour acidosis: from the passenger to the driver''s seat. Nat Rev Cancer, 2017. 17(10): p. 577-593.
19. Neri, D. and C.T. Supuran, Interfering with pH regulation in tumours as a therapeutic strategy. Nat Rev Drug Discov, 2011. 10(10): p. 767-77.
20. Vander Heiden, M.G., L.C. Cantley, and C.B. Thompson, Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 2009. 324(5930): p. 1029-33.
21. Helmlinger, G., et al., Acid production in glycolysis-impaired tumors provides new insights into tumor metabolism. Clin Cancer Res, 2002. 8(4): p. 1284-91.
22. Nakazawa, M.S., B. Keith, and M.C. Simon, Oxygen availability and metabolic adaptations. Nat Rev Cancer, 2016. 16(10): p. 663-73.
23. Corbet, C. and O. Feron, Cancer cell metabolism and mitochondria: Nutrient plasticity for TCA cycle fueling. Biochim Biophys Acta Rev Cancer, 2017. 1868(1): p. 7-15.
24. Draoui, N. and O. Feron, Lactate shuttles at a glance: from physiological paradigms to anti-cancer treatments. Dis Model Mech, 2011. 4(6): p. 727-32.
25. Khacho, M., et al., Acidosis overrides oxygen deprivation to maintain mitochondrial function and cell survival. Nature Communications, 2014. 5: p. 3550.
26. Corbet, C., et al., Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation. Cell Metab, 2016. 24(2): p. 311-23.
27. Lamonte, G., et al., Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress. Cancer Metab, 2013. 1(1): p. 23.
28. Peppicelli, S., F. Bianchini, and L. Calorini, Extracellular acidity, a "reappreciated" trait of tumor environment driving malignancy: perspectives in diagnosis and therapy. Cancer Metastasis Rev, 2014. 33(2-3): p. 823-32.
29. Parks, S.K., J. Chiche, and J. Pouyssegur, Disrupting proton dynamics and energy metabolism for cancer therapy. Nat Rev Cancer, 2013. 13(9): p. 611-23.
30. Wojtkowiak, J.W., et al., Chronic autophagy is a cellular adaptation to tumor acidic pH microenvironments. Cancer Res, 2012. 72(16): p. 3938-47.
31. Marino, M.L., et al., Autophagy is a protective mechanism for human melanoma cells under acidic stress. J Biol Chem, 2012. 287(36): p. 30664-76.
32. Fischer, K., et al., Inhibitory effect of tumor cell-derived lactic acid on human T cells. Blood, 2007. 109(9): p. 3812-9.
33. Dietl, K., et al., Lactic acid and acidification inhibit TNF secretion and glycolysis of human monocytes. J Immunol, 2010. 184(3): p. 1200-9.
34. Ciccone, V., et al., Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1alpha/VEGF signalling in MCF-7 breast cancer cells. J Exp Clin Cancer Res, 2018. 37(1): p. 311.
35. Maniotis, A.J., et al., Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol, 1999. 155(3): p. 739-52.
36. Ricci-Vitiani, L., et al., Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature, 2010. 468(7325): p. 824-8.