臺灣博碩士論文加值系統

Glioblastoma multiforme (GBM) is one the most lethal types of human brain cancers with high recurrence rate and resistance to conventional therapies due to its heterogeneous nature. Cancer immunotherapy has become a highlight in recent decade and inhibition of immune checkpoints has been shown to have successful outcomes in several cancers. In this present study, expression of CD86 was found to be regulated by TMZ treatment and radiation, and increased in a time-dependent manner in 05MG, U-87 MG and GL261 cells. Western blotting, IF and flow cytometry data demonstrated that inhibition of PI3K/AKT pathway by different inhibitors (LY294002, MK-2206 and PDTC) down-regulated the expression of CD86 induced by the treatments in 05MG cells. Notably, IF results also displayed a change in localization of CD86 expression from the cytoplasm at 0h to the cell surface membrane at 16h while addition of inhibitors reversed the phenomenon. In addition, relative proliferation rate of CD86 overexpressed 05MG cells was found to be greater as compared to that of the control. Cell surface expression of CD86 induced by TMZ treatment was also found to affect and elevate the cell surface CTLA-4 immune marker on Jurkat T cells. Besides, increase of CD86 expression was also observed in animal model under TMZ treatment. Therapy using TMZ along with anti-CTLA-4 antibody reduced the expression of CD86 and hampered tumor growth in GL261 syngeneic mouse model without compromising the physiological state of the mice. Taken together, this study demonstrates resistance of GBM to chemotherapy and radiotherapy may be attributed to the expression of CD86 through promotion of survival, growth and evasion from normal immune attack.

Contents
Acknowledgement i
Abstract iii
Contents iiii
List of Figures v
Chapter 1: Introduction 1
1.1 Glioblastoma multiforme (GBM) 2
1.2 Cancer immunotherapy 4
1.3 Immunomodulation in cancers and GBM 6
1.4 Immune checkpoints in GBM 8
Chapter 2: Motivation and Study Rationale 9
Chapter 3: Materials and Methods 10
3.1 Cell lines and cell culture 11
3.1.1 Cell lines 12
3.1.2 Cell culture 12
3.2 Temozolomide® (TMZ) treatment 12
3.2.1 Preparation of TMZ drug 12
3.2.2 Determination of IC50 13
3.2.3 TMZ treatment on GBM cancer cell lines 13
3.3 Radiation treatment 13
3.4 PI3K/AKT pathway inhibtion treatment 13
3.5 Gene delivery 14
3.6 Co-culture experiment 14
3.7 Reverse transcription polymerase chain reaction (RT-PCR) 14
3.8 Quantitative real time polymerase chain reaction (qRT-PCR) 15
3.9 Total cellular protein extraction 15
3.10 Western blotting 15
3.11 MTT cell viability/proliferation assay 16
3.12 Immunofluorescence (IF) staining 16
3.13 Flow cytometry 17
3.14 Animal model study 17
3.14.1 Generation of luciferase and GFP expressing GL261 17
3.14.2 GL261-Luc-GFP orthotopic transplantation and non-invasive imaging 17
3.14.2 Tail-vein injection (TVI) of drugs 18
3.15 Data analysis 18
Chapter 4: Results 19
4.1 Expression of CD86 is regulated by TMZ treatment in a time-dependent manner 20
4.2 Expression of CD86 is regulated by radiation treatment in a prompt and transient manner 20
4.3 Expression of CD86 is mediated through PI3K/AKT pathway 21
4.4 Overexpression of CD86 promotes cell proliferation in 05MG and enhances expression of CTLA-4 in Jurkat T cells 23
4.5 Combined therapy reduces the expression of CD86 and halts tumor growth in GL261 syngeneic mouse model 24
Chapter 5: Discussion and Conclusion 26
Chapter 6: References 29
Chapter 7: Figures 35
Chapter 8: Appendices 58

List of Figures
Figure 1: IC50 of 05MG, U-87 MG and GL261 cells. 36
Figure 2: Induction of CD86 and PD-L1 expressions in 05MG by TMZ treatment. 37
Figure 3: Induction of CD86 and PD-L1 expressions in U-87 MG by TMZ treatment. 38
Figure 4: Induction of CD86 and PD-L1 expressions in GL261 by TMZ treatment 39
Figure 5: Induction of CD86 and PD-L1 expressions in 05MG by radiation treatment. 40
Figure 6: Induction of CD86 and PD-L1 expressions in U-87 MG by radiation treatment. 41
Figure 7: Induction of CD86 and PD-L1 expressions in GL261 by radiation treatment. 42
Figure 8: Activation of PI3K/AKT pathway in 05MG under different treatments. 43
Figure 9: Activation of PI3K/AKT pathway in U-87 MG under different treatments. 44
Figure 10: Activation of PI3K/AKT pathway in GL261 under different treatments. 45
Figure 11: Inhibition of PI3K/AKT pathway by different inhibitors in 05MG. 46
Figure 12: TMZ treatment changed location of CD86 expression from the cytoplasm to the plasma membrane and addition of PI3K/AKT pathway inhibitors reversed the phenomenon and reduced CD86 expression level in 05MG. 47
Figure 13: MK-2206 reduced the cell surface expression of CD86 on 05MG. 49
Figure 14: PDTC reduced the cell surface expression of CD86 on 05MG. 50
Figure 15: Overexpression of CD86 enhanced 05MG proliferation rate. 51
Figure 16: CD86 induced by TMZ treatment on 05MG increased CTLA-4 mRNA level in Jurkat T cells. 52
Figure 17: Prior preparation of syngeneic animal model study. 53
Figure 18: Presence of tumor infiltrating CD3+CTLA-4+ T cells and Ki67+CD86+ GL261 tumor cells. 54
Figure 19: Drug treatment groups hampered tumor growth in vivo. 56
Figure 20: Combined therapy reduced the cell surface expression of CD86 on GL261 tumor cells in vivo. 57

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