神經膠質瘤是臨床中最難治癒的腦瘤，接受過治療的病人之存活年限依然低於兩年，目前治療方式以手術切除，依據惡性程度搭配放射治療及化療藥物，其中化療的第二線藥物為順鉑(Cisplatin)，順鉑目前廣用於各式腫瘤，然而其機制簡單，在腫瘤之中易產生抗性，尤其於缺氧區內，藥物更難以到達，因此解決缺氧區腫瘤對於順鉑的抗性在臨床研究中其中一個重要目標。本篇使用一合成新藥物Cisplatin analogue應用於小鼠星狀膠質瘤ALTS1C1，將Cisplatin經過修飾後，希望能夠標的於腫瘤缺氧區，並藉以分子中Cisplatin的機制加以毒殺細胞，並和Cisplatin做一比較。研究結果顯示於細胞實驗中，此兩種藥物正常氧氣濃度極缺氧環境下的細胞毒殺能力並無太大差異；在動物實驗中，於腫瘤生長前期治療時，Cisplatin治療後的缺氧區顯著下降，血管成熟度也較高，而Cisplatin analogue治療後的血管密度及缺氧區大小和控制組比較並無明顯差異，顯示出此兩種藥物對於腫瘤內的微環境有不同的影響，但Cisplatin analogue於缺氧區內有較高程度的細胞凋亡，表示出藥物有能力往缺氧區浸潤並毒殺細胞。在腫瘤前期結合Cisplatin和其analogue的治療中，顯示出此兩藥物有互補的功能，能夠更有效減緩腫瘤的生長速度。

Glioma is the most difficult brain tumors to be cured in clinic. Despite receiving treatment, the survival days are still below two years. After performing the maximal surgical resection, the best strategy is always to combine with radiotherapy or chemotherapy, which depend on the malignancy and phenotype. The frequently used second-line of chemotherapeutic drug is cisplatin, widely used against carcinomas, germ cell tumors, lymphomas, and sarcomas. It has a simple mechanism dependent on interfering with DNA replication and inducing apoptosis. However, the drug resistance in tumor hypoxia, where the drug is difficult to reach, is one of problem need to be solved. In this study, we used a cisplatin analogue derived from the modification of cisplatin in order to target the hypoxia cells of a murine astrocytoma, ALTS1C1. According to the MTT assay, we found that the cytotoxicity of cisplatin and cisplatin analogue was slightly decreased under hypoxia. In animal studies, the administration of cisplatin changed the microvascular density and hypoxia percentage in early stage but cisplatin analogue didn’t. However, the administration of cisplatin analogue significantly increased the apoptotic cells in hypoxic area. In addition, this study also found that the combination of cisplatin with cisplatin analogue are more effective than single treatment to slow down the tumor growth, indicating that cisplatin analogue may have different working targets to its parental compound.

摘要 I
Abstract II
誌謝 III
目次 IV
壹、緒論 1
1.1 惡性神經膠質瘤(Glioma) 1
1.2 腫瘤微環境 2
1.2.1 腫瘤缺氧區(Hypoxia area) 3
1.2.2 腫瘤新生血管(Tumor Angiogenesis) 4
1.3 順鉑(Cisplatin) 5
1.4 缺氧偵測試劑(Pimonidazole, PIMO) 5
1.5 實驗目的 6
貳、材料與方法 7
2.1 細胞實驗 7
2.1.1 配製細胞培養液DMEM 7
2.1.2 調配細胞培養液 7
2.1.3 細胞培養與繼代 7
2.1.4 藥物對ALTS1C1細胞的敏感性試驗 8
2.1.5 細胞存活率測試(MTT assay) 8
2.2 動物實驗 9
2.2.1 動物來源 9
2.2.2 動物分組 9
2.2.3 植入腫瘤細胞株 10
2.2.4 藥物治療 10
2.2.5 腫瘤犧牲與包埋儲存 11
2.2.6 腫瘤組織切片 11
2.2.7 腫瘤切片分析 14
參、實驗結果 16
3.1 ALTS1C1細胞對於化療藥物的敏感性測試 16
3.2 檢測藥物在動物腫瘤內的分佈 17
3.2.1 腫瘤生長曲線圖 17
3.2.2 腫瘤內藥物的分佈 17
3.2.3 比較缺氧區域細胞凋亡的程度 18
3.2.4 比較治療與否之微環境的差異 18
3.3 一般腫瘤治療模式之效果 19
3.3.1 腫瘤治療生長曲線圖 19
3.3.2 比較不同藥物對於腫瘤微環境的差異 20
3.3.3 腫瘤內藥物鉑與缺氧區之對照 20
3.4腫瘤於前期治療之效果 21
3.4.1 腫瘤前期治療生長曲線圖 21
3.4.2比較不同藥物對於腫瘤微環境的差異 22
3.4.3腫瘤內藥物鉑與缺氧區之對照 22
3.4.4 比較缺氧區域細胞凋亡的程度 23
肆、討論 24
伍、圖表 31
陸、參考資料 45

1. Vigneswaran, K., S. Neill, and C.G. Hadjipanayis, Beyond the World Health Organization grading of infiltrating gliomas: advances in the molecular genetics of glioma classification. Ann Transl Med, 2015. 3(7): p. 95.
2. Louis, D.N., et al., The 2007 WHO classification of tumours of the central nervous system. Acta …, 2007.
3. Nikiforova, M.N. and R.L. Hamilton, Molecular diagnostics of gliomas. Archives of pathology & …, 2011.
4. Stupp, R., et al., High-grade glioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO, 2014. 25 Suppl 3: p. 101.
5. Chen, R., A.L. Cohen, and H. Colman, Targeted Therapeutics in Patients With High-Grade Gliomas: Past, Present, and Future. Current treatment options in oncology, 2016. 17(8): p. 42.
6. Nieder, C., et al., Therapeutic options for recurrent high-grade glioma in adult patients: recent advances. Critical reviews in oncology/hematology, 2006. 60(3): p. 181-193.
7. Wang, S.-C., et al., Tumor-secreted SDF-1 promotes glioma invasiveness and TAM tropism toward hypoxia in a murine astrocytoma model. Laboratory Investigation, 2011. 92(1): p. 151-162.
8. Balkwill, F.R., M. Capasso, and T. Hagemann, The tumor microenvironment at a glance. J Cell Sci, 2012.
9. Dewhirst, M.W., et al., Multiple Etiologies of Tumor Hypoxia Require Multifaceted Solutions. Clinical Cancer Research, 2007. 13(2): p. 375-377.
10. Thomas, S.N., et al., Exosomal proteome profiling: a potential multi-marker cellular phenotyping tool to characterize hypoxia-induced radiation resistance in breast cancer. Proteomes, 2013. 1(2): p. 87-108.
11. Semenza, G.L., Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology, 2004.
12. Gilkes, D.M., G.L. Semenza, and D. Wirtz, Hypoxia and the extracellular matrix: drivers of tumour metastasis. Nature Reviews Cancer, 2014. 14(6): p. 430-439.
13. Covello, K.L., et al., HIF-2 regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes & development, 2006. 20(5): p. 557-570.
14. Keith, B., R.S. Johnson, and C.M. Simon, HIF1α and HIF2α: sibling rivalry in hypoxic tumour growth and progression. Nature Reviews Cancer, 2011.
15. Gray, L.H., et al., The Concentration of Oxygen Dissolved in Tissues at the Time of Irradiation as a Factor in Radiotherapy. The British Journal of Radiology, 1953. 26(312): p. 638-648.
16. Comerford, K.M., et al., Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer research, 2002.
17. Bayer, C. and P. Vaupel, Acute versus chronic hypoxia in tumors. Strahlentherapie und Onkologie, 2012. 188(7): p. 616-627.
18. Rohwer, N. and T. Cramer, Hypoxia-mediated drug resistance: novel insights on the functional interaction of HIFs and cell death pathways. Drug Resistance Updates, 2011.
19. Sullivan, R., et al., Hypoxia-induced resistance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity. Molecular cancer therapeutics, 2008. 7(7): p. 1961-1973.
20. Shannon, A.M., et al., Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies. Cancer treatment reviews, 2003. 29(4): p. 297-307.
21. Wilson, W.R. and M.P. Hay, Targeting hypoxia in cancer therapy. Nature Reviews Cancer, 2011.
22. Folkman, J. and D. Hanahan. Switch to the angiogenic phenotype during tumorigenesis. in Princess Takamatsu Symposia. 1990.
23. Weis, S.M. and D.A. Cheresh, Tumor angiogenesis: molecular pathways and therapeutic targets. Nature Medicine, 2011. 17(11): p. 1359-1370.
24. Patan, S., Vasculogenesis and angiogenesis. Angiogenesis in brain tumors, 2004.
25. Hellberg, C., A. Ostman, and C.H.H. Heldin, PDGF and vessel maturation. Recent results in cancer research. Fortschritte der Krebsforschung. Progrès dans les recherches sur le cancer, 2010. 180: p. 103-114.
26. Stordal, B. and M. Davey, Understanding cisplatin resistance using cellular models. IUBMB life, 2007. 59(11): p. 696-699.
27. Eastman, A., The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Pharmacology & therapeutics, 1987. 34(2): p. 155-166.
28. Dasari, S. and P.B. Tchounwou, Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol, 2014. 740: p. 364-78.
29. Siddik, Z.H., Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene, 2003. 22(47): p. 7265-7279.
30. Rademakers, S.E., et al., Metabolic markers in relation to hypoxia; staining patterns and colocalization of pimonidazole, HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4. BMC Cancer, 2011.
31. Airley, R.E., et al., GLUT‐1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: Relationship to pimonidazole binding. International Journal of Cancer, 2003: p. 85-91.
32. Varia, M.A., D.P. Calkins-Adams, and L.H. Rinker, Pimonidazole: a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in cervical carcinoma. Gynecologic …, 1998.
33. Jubb, A.M., F.M. Buffa, and A.L. Harris, Assessment of tumour hypoxia for prediction of response to therapy and cancer prognosis. Journal of cellular and …, 2010.
34. Chekenya, M., et al., The glial precursor proteoglycan, NG2, is expressed on tumour neovasculature by vascular pericytes in human malignant brain tumours. Neuropathology and Applied Neurobiology, 2002. 28(5): p. 367-380.
35. Raza, A., M.J. Franklin, and A.Z. Dudek, Pericytes and vessel maturation during tumor angiogenesis and metastasis. American journal of …, 2010.
36. Jain, R.K., Molecular regulation of vessel maturation. Nature Medicine, 2003. 9(6): p. 685-693.
37. Charles, N.A., et al., The brain tumor microenvironment. Glia, 2012. 60(3): p. 502-514.
38. Jain, A., et al., Glioblastoma: Current Chemotherapeutic Status and Need for New Targets and Approaches.
39. Rocha, C.R.R., et al., Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo. Cell death & disease, 2014. 5.
40. Wohlkoenig, C., et al., Hypoxia-induced cisplatin resistance is reversible and growth rate independent in lung cancer cells. Cancer letters, 2011. 308(2): p. 134-143.
41. Tanabe, M., et al., Activating transcription factor 4 increases the cisplatin resistance of human cancer cell lines. Cancer research, 2003. 63(24): p. 8592-8595.
42. Wu, H.-M.M., et al., Hypoxia-induced autophagy mediates cisplatin resistance in lung cancer cells. Scientific reports, 2015. 5: p. 12291.
43. Rzymski, T., et al., Role of ATF4 in regulation of autophagy and resistance to drugs and hypoxia. Cell Cycle, 2009.
44. Rice, G.C., C. Hoy, and R.T. Schimke, Transient hypoxia enhances the frequency of dihydrofolate reductase gene amplification in Chinese hamster ovary cells. Proceedings of the National …, 1986.
45. Torti, S.V. and F.M. Torti, Iron and cancer: more ore to be mined. Nature reviews. Cancer, 2013. 13(5): p. 342-355.
46. De Palma, M., et al., Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer cell, 2005. 8(3): p. 211-226.
47. Huang, F.J., et al., Pericyte deficiencies lead to aberrant tumor vascularizaton in the brain of the NG2 null mouse. Developmental …, 2010.
48. Routes, E.A., DRUG ABSORPTION, DISTRIBUTION AND ELIMINATION; PHARMACOKINETICS, in DRUG ABSORPTION, DISTRIBUTION AND ELIMINATION; PHARMACOKINETICS. 2015.
49. Ruggeri, B.A., F. Camp, and S. Miknyoczki, Animal models of disease: pre-clinical animal models of cancer and their applications and utility in drug discovery. Biochemical pharmacology, 2014.