肺癌是全世界癌症死亡的首要原因。分子流行病學研究顯示，肺癌致癌基因存在差異。例如表皮生長因子受體（epidermal growth factor receptor，EGFR）發生突變的非小細胞肺癌（NSCLCs, non-small cell lung cancer）在亞洲人中的比例，比白人患者顯著較高。在台灣，特別是有關非吸煙女性患者的肺腺癌，EGFRL858R點突變發生頻率為最高。
之前研究顯示Forkhead box protein M1（Foxm1）在細胞增殖、分化，以及多種細胞類型的腫瘤發生中參與重要角色，是細胞中重要的轉錄因子。最近透過基因轉殖小鼠模式的研究發現，Foxm1在致癌物及致癌基因誘發肺癌癌化過程上扮演重要角色。在此，我們透過基因工程小鼠（Genetical Engineered Mouse Model）肺癌模式，條件性基因剔除老鼠肺部細支氣管上Club 細胞中的Foxm1基因（CCSP/EGFRL858R/Foxm1-/-），發現剔除Foxm1 會使EGFR誘發的腫瘤及增生病變顯著減少；同時，以微米級電腦斷層掃描造影系統(micro computed tomography, microCT)分析肺內部結構，以及肺臟石蠟切片作H＆E染色病理學檢測，發現剔除Foxm1組中明顯減少點突變EGFRL858R誘發之腫瘤數目。此外，我們也發現，於氣管內注射表現CRE的腺病毒（Ad-Cre）以誘發Foxm1基因剔除，可使致癌EGFR所誘發的肺腫瘤萎縮（CCSP/EGFRL858R/Foxm1fl/fl）。我們的實驗結果證明，Foxm1在EGFR致癌突變所引起之肺部腫瘤發生中至關重要。

Lung cancer is the leading cause of cancer death worldwide. Many driver mutations for lung cancer have been identified and recent research showed that the spectrum of these oncogenic mutations is variable among racial groups. Molecular epidemiology of mutations in lung cancers showed that prevalence of EGFR oncogenic mutations in Asian patients with non-small cell lung cancers (NSCLCs) is remarkably higher than Caucasian patients. In Taiwan, EGFRL858R missense mutation is predominant found in lung adenocarcinoma (ADC), especially for non-smoker women patients.
Transcription factor Forkhead Box m1 (Foxm1) plays important roles in cellular proliferation, differentiation and tumorigenesis of multiple cell types. Previous research showed that Foxm1 is highly expressed in human lung cancers and we have demonstrated that Foxm1 is required for the initiation of lung tumorigenesis induced by carcinogens or genetically induced lung cancer models. Herein, we are investigating whether Foxm1 is involved in oncogenic EGFRL858R mutation induced lung ADC. We used a genetically engineered mouse model that renders conditional deletion of Foxm1 gene in lung Club cells. The result showed that knockout Foxm1 in airway epithelial cells caused a striking reduction in the size of lesions in EGFRL858R expressing lungs as detected by the pre-clinical micro-computed tomography (microCT), which is consistent with the results from H&E-stained lung pathological examination of cancer. We demonstrated that deletion of Foxm1 prevented the initiation of lung tumors by oncogenic EGFRL858R in lung ADC. Additionally, intratracheal injections of Cre expressing adenovirus (Ad-Cre) to mice with pre-existed EGFR mutated lung tumors caused lung tumor regression. Our results demonstrated that Foxm1 is critical for EGFRL858R -mediated lung tumorigenesis.

中文摘要 - 1 -
ABSTRACT - 2 -
ABBREVIATIONS - 3 -
1. INTRODUCTION - 11 -
1.1 Lung cancer and gene mutation-related lung adenocarcinoma - 11 -
1.2 Roles of Foxm1 related with carcinogenesis - 13 -
1.3 Signal pathway related with EGFR - 15 -
1.4 Mouse models for lung cancer research - 16 -
1.5 MicroCT technique for the study of mouse model - 18 -
1.6 Adenovirus expressing Cre-recombinase (Ad-Cre) and intratracheal injection for gene knockout - 20 -
2. OBJECTIVE OF THIS STUDY - 22 -
3. MATERIAL AND METHODS: - 23 -
3.1 Mouse preparation - 23 -
3.1.1 List of mouse source - 23 -
3.1.2 Quadruple tangencies mice model - 23 -
3.1.3 Keeping and breeding mice - 23 -
3.1.4 Breeding strategy of transgenic mice for conditional gene activation using inducible Cre mice - 24 -
3.1.5 Mouse genomic DNA extraction and genotyping - 24 -
3.2 MicroCT procedures - 27 -
3.2.1 Preparation of micro-CT imaging - 27 -
3.2.2 MicroCT image acquisition - 27 -
3.2.3 MicroCT image analysis - 28 -
3.3 processing of mouse tissue samples - 28 -
3.3.1 Harvesting mouse lungs - 28 -
3.3.1 Total lung RNA extraction - 29 -
3.3.2 Paraffin blocks embedding - 29 -
3.3.3 Hematoxylin and Eosin (H&E) staining - 29 -
3.3.4 Quantitative analysis mRNA expression by Real-time PCR - 29 -
3.4 Adenovirus infection - 30 -
3.4.1 Construction of Cre expression adenovirus - 30 -
3.4.2 Cell culture - 31 -
3.4.3 preparation of Recombinant Adenovirus - 31 -
3.4.4 Intratracheal injection for C/EGFR/Foxm1fl/fl transgenic mice - 32 -
3.5 Statistical analysis - 32 -
4. RESULT - 33 -
4.1 Generation of Doxycycline inducible Foxm1 expression in quadruple tangencies mice was switched on by doxycycline treatment. - 33 -
4.2 Inducible knock out Foxm1 prolong the surviving of the oncogenic EGFRL858R mutant lung cancer model mice - 34 -
4.3 Inducible knock out Foxm1 could significant induce the lung tissue tumor in the oncogenic EGFRL858R mutant lung cancer model by microCT image analysis - 35 -
4.4 Comparison of microCT image with histopathology shows the induction of tumorigenesis through the Foxm1 knock out by the Cre expression - 36 -
4.5 Lung adenocarcinoma induction of intratracheal injection adenovirus expressing Cre-recombinase by knock out Foxm1 expression - 36 -
5. DISCUSSION - 38 -
6. ACKNOWLEDGEMENTS - 43 -
7. REFERENCES - 44 -
8. FIGURE - 52 -
Figure 1. Schematic diagram of EGFRL858R transgenic mice conditional activation by Tet-On system - 52 -
Figure 2. The breeding strategy of conditional activation of EGFR and knockout Foxm1 through Tet-on system - 53 -
Figure 3. The Schematic of expression of Foxm1 knock out in EGFR mutant mice model - 54 -
Figure 4. Increase of weight in EGFR-mutated mouse lungs - 55 -
Figure 5. Kaplan–Meier survival curve for mouse expressing oncogenic EGFR w/ and w/o Foxm1 - 56 -
Figure 6. micro-computed tomography (microCT) shows a decrease of lung tumor loading in C/EGFR/Foxm1 KO mice with 3.5 month Dox diet - 57 -
Figure 7. micro-computed tomography (microCT) shows a decrease of lung tumor loading in C/EGFR/Foxm1 KO mice with 5 month Dox diet - 58 -
Figure 8. Quantitative analysis for microCT images of lung tumor loading in mice - 59 -
Figure 9. Percentage of soft tissue volume/ total lung volume in mice after Dox treatment for 3.5 months - 60 -
Figure 10. Percentage of soft tissue volume/ total lung volume in mice after Dox treatment for 5 months - 61 -
Figure 11. H&E staining of lungs for histopathological analysis in 3.5months Dox diet mice - 62 -
Figure 12. H&E staining of lungs for histopathological analysis in 5months Dox diet mice - 63 -
Figure 13. The RNA levels of C/EGFR/Foxm1fl/fl ,C/EGFR/Foxm1-/- in mouse lungs - 64 -
Figure 14. Schematic diagram of using Cre-recombinase expressing adenovirus (ad-Cre) to induce Foxm1 deletion in EGFR expressing lung tumor - 65 -
Figure 15. The procedure for Ad-Cre intratracheal injection (IT injection) and lung tumor microCT image analysis - 66 -
Figure 16. Decreased tumor loading in EGFRL858R mouse lungs after Ad-Cre IT treatment - 67 -
Figure 17. Model for EGFRL858R missense mutation related with Foxm1 expression - 68 -
Figure s1. The lung appearance of C/EGFR/Foxm1 fl/fl and C/EGFR/Foxm1-/- transgenic mice - 69 -
Figure s2. Increase of weight in EGFR-mutated mouse lungs with different gender - 70 -
Figure s3. Kaplan–Meier survival curve for EGFR mutant male-mouse - 71 -
Figure s4. Kaplan–Meier survival curve for EGFR mutant female-mouse - 72 -
Figure s5. Three-dimension of littermate control mouse treat with 3.5 months Dox diet - 73 -
Figure s6. Three-dimension of C/EGFR/Foxm1fl/fl mouse treat with 3.5 months Dox diet - 74 -
Figure s7. Three-dimension of C/EGFR/Foxm1-/- mouse treat with 3.5 months Dox diet - 75 -
Figure s8. Three-dimension of C/EGFR/Foxm1fl/fl mouse treat with 5 months Dox diet - 76 -
Figure s9. Three-dimension of C/EGFR/Foxm1-/- mouse treat with 5 months Dox diet - 77 -
Figure s10. Image captured by the microCT of littermate control with Dox 3.5 months. - 79 -
Figure s11. Image captured by the microCT of C/EGFR/Foxm1 fl/fl with Dox 3.5 months - 81 -
Figure s12. Image captured by the microCT of C/EGFR/Foxm1 -/- with Dox 3.5 months - 83 -
Figure s13. Image captured by the microCT of C/EGFR/Foxm1 fl/fl and C/EGFR/Foxm1 -/- with Dox 5 months - 85 -
Figure s14. Distribution of three EGFR mutant mice groups in Dox diet treatment 3.5 months - 86 -
Figure s15. Distribution of three groups EGFR mutant mice groups in Dox diet treatment 5 months - 87 -
Figure s16. The method of comparison of before/after Ad-Cre IT injection of mice - 88 -
9. TABLES - 89 -
Table 1. Full list of EGFR mutant mice model - 91 -
Table 2. Full list of analysis in mouse weight and lung weight - 92 -
Table 3. Full list of quantitative analysis in total lung volume and soft tissue volume - 94 -
10. APPENDIX - 95 -
Figure. a1: MicroCT Image Reconstruction and Voxels - 95 -
Figure. a2: Calculation of CT number - 96 -
Figure. a3: Hounsfield Unit Scale - 97 -

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