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dc.contributor.advisor | 成佳憲(Jason Chia-Hsien Cheng) | |
dc.contributor.author | Yun Chiang | en |
dc.contributor.author | 江韻 | zh_TW |
dc.date.accessioned | 2023-03-19T22:43:35Z | - |
dc.date.copyright | 2022-10-03 | |
dc.date.issued | 2022 | |
dc.date.submitted | 2022-08-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85099 | - |
dc.description.abstract | 放射線治療為主的膀胱保留療法是針對局部肌肉侵犯型的膀胱泌尿道腫瘤的手術替代治療,其結果和根治性膀胱切除術相當,但是僅適用於部分患者。而且臨床前研究顯示局部放射線對抑制膀胱癌細胞生長效果有限,甚至可能會刺激腫瘤轉移進展。即使在過去的十年中,免疫療法已顯示出對治療局部侵犯和轉移性膀胱癌的前景,膀胱癌對免疫檢查點抑制劑的反應率還是有限。著眼於近年來大量的臨床前研究證明放射線具有多種免疫調節功能,而且合併使用放射治療和免疫治療具有潛在的協同作用,釐清放射線照射後的腫瘤微環境對克服膀胱癌之放射線阻抗性的研究具有重要的臨床意義。本論文研究放射線照射後的腫瘤微環境中,膀胱癌細胞和免疫細胞之間的相互作用,以改善局部侵犯或轉移性膀胱癌的局部放射治療效果。 第一部分是研究放射線照射對於膀胱癌細胞的影響。我們發現在接受含有放射線治療的膀胱保留患者中,膀胱腫瘤內NFκB的免疫化學染色過度表現和較差的預後有關。進一步的細胞實驗顯示放射線照射會活化膀胱癌細胞的NFκB的訊息傳導,增強膀胱癌細胞的存活和轉移能力。 第二部分是研究放射線照射後的膀胱癌細胞對腫瘤微環境內免疫細胞的作用。我們的小鼠動物實驗顯示膀胱癌細胞株異體腫瘤局部放射線照射會增加遠端轉移的風險,且免疫組織分析呈現腫瘤相關巨噬細胞匯聚在放射線照射過後的腫瘤微環境。因此,我們透過細胞及小鼠動物實驗探索放射線照射過後的膀胱癌細胞對腫瘤相關巨噬細胞的影響和膀胱癌遠端轉移以及存活的相關性及機轉。我們的研究顯示膀胱癌細胞受到放射線刺激後透過活化NFκB的訊息傳導而分泌CCL2,而此細胞激素會促使骨髓分泌表現CCR2的腫瘤相關巨噬細胞匯聚在放射線照射過後的腫瘤微環境,同時趨化M1亞型腫瘤相關巨噬細胞成M2亞型腫瘤相關巨噬細胞,進一步造成放射線後的肺部轉移。抑制CCL2-CCR2的訊息傳導會降低放射線後肺部轉移的發生率。我們也發現在放射線照射過後的腫瘤微環境中的腫瘤相關巨噬細胞可能透過表現PD-L1來抑制毒殺性CD8+T細胞免疫功能的機制。 總結本論文發現,放射線藉由活化膀胱癌腫瘤細胞的NFκB訊息傳導使膀胱癌腫瘤細胞分泌CCL2,進一步透過CCL2的細胞激素反應影響腫瘤相關巨噬細胞的招募極化,造成放射線抗性和增加遠端轉移的風險。藉由發展及合併使用相關的抑制劑,我們就有機會改善放射線治療在膀胱癌的療效及預後。 | zh_TW |
dc.description.abstract | Radiotherapy (RT)-based bladder preservation therapy is an alternative treatment to surgery for locally advanced muscle-invasive bladder cancer (MIBC). It results in comparable outcome to radical cystectomy, but only for selected patients. In preclinical studies, RT contributes to limited suppression of tumor growth and might stimulate metastatic cascade of bladder cancer cells. Immunotherapy based on immune checkpoint inhibitors (ICIs) has provided promises in the past decade for the treatment of locally advanced and metastatic MIBC, albeit with a modest objective response. With many pre-clinical data demonstrating multiple immunomodulatory functions of RT and the potential synergistic effect of a combinational use of RT and immunotherapy, the exploration of postirradiated tumor microenvironment (TME) to overcome the radioresistance in MIBC will be of great clinical significance. This thesis addresses the interactions between bladder cancer cells and immune cells in the postirradiated TME in order to improve treatment outcomes of locally advanced or metastatic MIBC treated with local RT. The first part of this thesis addresses the effect of RT on bladder cancer cells. We found increased immunoreactivity of nuclear factor-kappa B (NFκB) by immunohistochemical staining in patients receiving RT-based bladder-preserving therapy was associated with significantly unfavorable outcomes. Further in vitro experiments demonstrated up-regulation of NFκB signaling after RT contributed to enhanced proliferative and invasive capability of bladder cancer cells. The second part of the thesis focuses on the influence of irradiated bladder cancer cells on the immune cells in the TME. Our in vivo work demonstrated the increased risk of distant lung metastasis after local RT to the primary tumors using ectopic allograft C57BL/6 mouse model with murine MB49 bladder cancer cell line. We also found the increased infiltration of tumor-associated macrophages (TAMs) in the postirradiated TME by immunohistochemical analysis. Therefore, we explored the association between irradiated bladder cancer cells and TAMs and their impact on survivals and metastatic capability of bladder cancer using in vitro and in vivo experiments. Our study demonstrated that bladder cancer cells responded to RT by producing C-C motif ligand 2 (CCL2) through NFκB signaling, as well as recruited TAMs presented with chemokine receptor 2 (CCR2) from bone marrow and polarized M1-type TAMs toward M2-type TAMs. This phenotypic TAM transformation promoted the pulmonary metastasis of bladder cancer cells after RT. Blockade of the CCL2-CCR2 axis reduced post-RT pulmonary metastasis. We also showed that TAMs in the post-RT TME may subvert CD8+ T cell immune surveillance by expressing PD-L1. The thesis concludes that RT-activated NFκB signaling in bladder cancer cells with secretion of CCL2, which contributes to recruitment and polarization of TAMs, resulting in radioresistance and risk of distant metastasis. With the development and combination of the corresponding inhibitors, we may be able to enhance the response and improve the outcome of RT-based treatment in bladder cancer. | en |
dc.description.provenance | Made available in DSpace on 2023-03-19T22:43:35Z (GMT). No. of bitstreams: 1 U0001-0908202217265900.pdf: 3859727 bytes, checksum: d1e8fd0592758c081e6b7a48f6fd463c (MD5) Previous issue date: 2022 | en |
dc.description.tableofcontents | Table of contents 口試委員審定書 i 誠摯感謝 ii 中文摘要 iii Abstract v Table of contents vii List of Figures ix Chapter 1. Introduction 1 1.1 Radiotherapy in bladder cancer 1 1.2 The association between radiotherapy and NFκB signaling in bladder cancer 2 1.3 Immunotherapy in bladder cancer 2 1.4 TAMs and postirradiated TME of bladder cancer 3 1.5 CCL2-CCR2 chemokine axis and bladder cancer 5 1.6 Interaction of TAMs and cytotoxic T cell 6 Chapter 2. Methods and Materials 7 2.1 Cell lines and cultures 7 2.2 Reagents 8 2.3 In vitro radiation treatment 8 2.4 In vitro Transwell coculture system 8 2.5 Evaluation and interpretation of immunohistochemistry (IHC) 9 2.6 Cytokine assay analysis 9 2.7 Enzyme-lined immunosorbent assay (ELISA) and preparation of mouse serum samples for ELISA 10 2.8 Colony formation assay 11 2.9 Cell invasion assay 11 2.10 Real-time quantitative reverse transcription (qRT-PCR) 12 2.11 FACS analysis 12 2.12 Western blot assay 13 2.13 Immunofluorescence staining 13 2.14 In vivo ectopic allograft mouse model, tail vein mouse model and radiation treatment 14 2.15 Statistical analysis 14 Chapter 3. Results 15 3.1 NFκB overexpression is a negative prognosticator in MIBC patients undergoing TMT 15 3.2 Radiotherapy-induced NFκB activation is associated with invasiveness and proliferation of bladder cancer cells 15 3.3 Local radiotherapy delays primary ectopic allograft tumor growth but promotes pulmonary metastasis in vivo 15 3.4 Bladder cancer cells interact with M1-type TAMs and respond to radiotherapy by secretiing CCL2 in vitro 16 3.5 Radiotherapy-induced NFκB activation contributes to CCL2 secretion of bladder cancer cells in vitro 17 3.6 CCL2 concentration in mouse serum is increased after radiotherapy 17 3.7 Bladder tumor-derived CCL2 recruits TAMs 18 3.8 Bladder tumor-derived CCL2 increases the infiltration of M2-type TAMs 18 3.9 Bladder tumor-derived CCL2 induces the phenotyptic transformation of TAMs 18 3.10 CCL2-CCR2 activation enhances the protumor effects of M2-type TAMs 19 3.11 CCL2-CCR2 activation has no impact on survival of bladder cancer cells 20 3.12 Inhibition of CCL2-CCR2 activation impedes the recruitment of TAMs 20 3.13 Inhibition of CCL2-CCR2 activation hinders the phenotypic transformation of TAMs 21 3.14 Inhibition of CCL2-CCR2 activation suppresses pulmonary metastasis but not local tumor conotrol 21 3.15 Higher expression of M2-type TAMs is observed in the human bladder tumor 21 3.16 Interaction of TAMs and T cells 21 Chapter 4. Discussion 22 4.1 TAMs and radioresistance of bladder cancer 22 4.2 NFκB signaling in bladder cancer 23 4.3 CCL2-CCR2 axis in bladder cancer 24 4.4 The influence of TAMs on adaptive immunity 25 4.5 Limitations and future work 26 Chapter 5. Conclusion 27 Figures 28 References 63 Appendix 67 List of Figures Figure 1. Positive NFκB staining is associated with lower rates of survivals in patients treated with trimodality bladder-preserving therapy. 28 Figure 2. Immunohistochemical staining of NFκB in bladder tumors. 30 Figure 3. Irradiation induced up-regulation of NFκB signaling, contributing to the proliferative capability of bladder cancer cells. 31 Figure 4. Immunofluorescence staining of phospho-p65 after radiotherapy of MB49 vector-control and MB49 NFκB knock-down cells. 32 Figure 5. Cell invasion assy of MB49 vector-control and MB49 NFκB knock-down cells treated without and with radiotherapy. 33 Figure 6. The workflow of the in vivo experiment. 34 Figure 7. Representative images of primary tumors on fluoroscopic scans and gross tumor sections and the tumor growth curves of the radiotherapy (RT) group and no RT group. 35 Figure 8. Representative images and numbers of metastatic lung tumors on cone-beam CT scans and sets of lung tissues with counted gross surface metastases of the radiotherapy (RT) and no RT group. 36 Figure 9. Cytokine array analysis of the cultured medium with MB49 bladder cancer cells after 0-Gy or 5-Gy radiotherapy coculture with or without M1-type RAW 264.7 cells. 37 Figure 10. Mouse Cytokine Array to detect multiple cytokines and the expression of multiple chemokines, growth factors, and other proteins in the cultured medium samples. 38 Figure 11. MB49 bladder cancer cells were treated with 0-Gy or 5-Gy irradiation and then cultured alone or cocultured with M1-type RAW 264.7 cells, followed by ELISA and qRT–PCR to detect CCL2 expression. 39 Figure 12. MB49 bladder cancer cells were treated with 0-Gy or 5-Gy irradiation and then cultured alone or cocultured with M2-type RAW 264.7 cells, followed by ELISA and qRT–PCR to detect CCL2 expression. 40 Figure 13. MB49 bladder cancer cells were treated with 0-Gy or 5-Gy irradiation and then cultured alone or cocultured with M1-type or M2-type bone marrow-derived macrophages, followed by ELISA to detect CCL2 expression. 41 Figure 14. UMUC3 bladder cancer cells were treated with 0-Gy or 5-Gy irradiation and then cultured alone or cocultured with M1-type or M2-type THP-1 cells, followed by ELISA to detect CCL2 expression. 42 Figure 15. MBT2 bladder cancer cells were treated with 0-Gy or 5-Gy irradiation and then cultured alone or cocultured with M1-type or M2-type Raw 264.7 cells, followed by ELISA to detect CCL2 expression. 43 Figure 16. RT–PCR analyses of CCL2 and CCR2 expression in nonirradiated and irradiated MB49 bladder cancer cells. 44 Figure 17. ELISA and qRT–PCR were used to detect CCL2 expression of MB49 bladder cancer cells (0-Gy or 5-Gy) with or without NFκB inhibitor. 45 Figure 18. MB49 bladder cancer cells were treated with 0-Gy or 5-Gy irradiation and then cultured alone or cocultured with M1-type or M2-type RAW 264.7 cells, followed by qRT–PCR to detect NFκB expression. 46 Figure 19. Serum samples of mice in the no radiotherapy (RT) groups and RT groups were obtained by submandibular blood collection and the expression of CCL2 was evaluated by ELISA. 47 Figure 20. Representative images of surface metastases of lungs and metastatic lung tumors on cone-beam CT scans of tail vein mouse model, and the expression of CCL2 of serum samples was evaluated by ELISA. 48 Figure 21. Cell invasion assy of RAW 264.7 cells (M0-type, M1-type, and M2-type). 49 Figure 22. Representative microscopic images on immunohistochemically stained primary tumor tissue sections, lung tissue sections, and metastatic pulmonary tumor sections with CD206, CD68 and CD86. 50 Figure 23. qRT–PCR analysis of M1-type RAW 264.7 cells. 51 Figure 24. Cell invasion assy of MB49 bladder cancer cells. 53 Figure 25. Colony formation assays of irradiated MB49 bladder cancer cells with or without coculture with M1-type RAW 264.7 or M2-type RAW 264.7 cells or with additional recombinant mouse CCL2 protein. 54 Figure 26. The mRNA expression of CCR2 was analyzed by qRT-PCR in the primary tumor, and the percentage of CD11b+/CCR2+ cells was analyzed by flow cytometry analysis in the primary tumors and lung tissues. 55 Figure 27. A representative flow cytometry analysis showed the percentage of CD11b+/CCR2+ cells in the metastatic lung tissues in the tail vein mouse model. 56 Figure 28. The gene expression of markers of M1-type TAMs and M2-type TAMs in primary tumors was evaluated by qRT–PCR. 57 Figure 29. The tumor growth curves in the control group, RT group, INCB3344 group , and INCB3344 plus RT group. 58 Figure 30. Representative images and numbers of gross surface metastases of lungs, and metastatic lung tumors on cone-beam CT scans from each treatment group. 59 Figure 31. Representative microscopic images the number of cells with positive staining are shown on immunohistochemically stained bladder tumor tissue sections before and during or after radiotherapy-based trimodality treatment with CD206, CD68 and CCL2. 60 Figure 32. PD-L1 expression of M1-type RAW 264.7 cells in vitro, the gene expression levels of PD-L1 in the primary tumors, and the percentage of CD11b+/PD-L1+ cells in the primary tumors of radiotherapy (RT) group and no RT group are shown. 61 Figure 33. A graphic summary of the main findings presented in the study is illustrated with BioRender.com. 62 | |
dc.language.iso | en | |
dc.title | 探索NFκB引發的CCL2訊息傳導對放射線照射後的膀胱腫瘤微環境中巨噬細胞極化及療效的機轉 | zh_TW |
dc.title | Exploring the mechanism of NFκB-induced CCL2 signaling on polarization of macrophages and radiotherapeutic effect in the post-irradiated bladder cancer microenvironment | en |
dc.type | Thesis | |
dc.date.schoolyear | 110-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 徐志宏(Chih-Hung Hsu),闕士傑(Jeff Shih-Chieh Chueh),陳妙芬(Miao-Fen Chen),黃正仲(Jeng-Jong Hwang) | |
dc.subject.keyword | 膀胱癌,腫瘤相關巨噬細胞,放射線治療,腫瘤微環境,NFκB,CCL2, | zh_TW |
dc.subject.keyword | Bladder cancer,Tumor-associated macrophages,Radiation,Tumor microenvironment,NFκB,CCL2, | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU202202219 | |
dc.rights.note | 同意授權(限校園內公開) | |
dc.date.accepted | 2022-08-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 腫瘤醫學研究所 | zh_TW |
dc.date.embargo-lift | 2022-10-03 | - |
Appears in Collections: | 腫瘤醫學研究所 |
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