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

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。

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.