星形胶质细胞分泌CCL2对复发性髓母细胞瘤干性维持的机制研究

Mortality among the medulloblastoma patients is rarely due to the primary tumor, but rather mainly due to the recurrence and dissemination. Although there exist currently no therapies approved or clinical trials for the specific treatment of medulloblastoma relapses, clearly the recurrent compartment should be the focus of research in order to decrease the morbidity. We previously found that both medulloblastoma stem-like cells (MBSCs) and tumor-associated astrocytes (TAA) promoted the malignant progression. Furthermore, our findings presented that the proportion of MBSCs significantly elevated because of the resistance to chemo-/radiotherapy and TAA still highly distributed contributing to the self-renewal of MBSCs through CCL2 secretion in relapsed medulloblastoma. However, the underlying molecular mechanism of stemness maintenance and the pathways activated in MBSCs to drive recurrence and metastases remains illustrative. Meanwhile, targeting the CCL2 signaling made significant sense to attenuate the Notch pathway induced stem-like properties in relapsed MBSCs. Therefore, it is supposed that the stemness maintenance of relapsed medulloblastoma depends in part on the TAA-derived high level of CCL2. Based on our previous results, we will further detect the relapse-promoting effects of TAA-secreted CCL2 by utilizing the recurrent medulloblastoma xenografts and chemotherapy-induced relapsed models of transgenic mice, as well as investigate the mechanistic pattern of stem-like properties regarding the CCL2/CCR2 interplaying with Notch signaling during recurrence, which provides evidence for the novel target identification in recurrent tumor micro-environment.

目前复发性髓母细胞瘤仍缺乏有效治疗手段，患者预后不良，且肿瘤复发播散的机制尚不明确。我们前期研究发现髓母细胞瘤干细胞及星形胶质细胞在肿瘤进展中发挥重要作用。常规治疗虽可杀灭部分肿瘤细胞，但肿瘤干细胞因耐药性强而在复发播散时进一步富集，星形胶质细胞对放化疗不敏感仍广泛浸润，其释放CCL2等因子促进肿瘤干细胞自我更新，但CCL2调控肿瘤干性的分子机理并不清楚，其在髓母细胞瘤复发播散中的作用还未有研究。我们检测患者放化疗复发标本时，发现Notch通路在介导CCL2维持肿瘤干性及促进复发播散中至关重要。因此，我们将采用人髓母细胞瘤移植瘤模型及化疗诱导转基因小鼠复发模型，继续研究复发性肿瘤炎性微环境中星形胶质细胞的活化状态及维持肿瘤干性的因素，阐明CCL2协同Notch通路调控肿瘤干性进而促进复发播散的分子机制，评价干预CCL2/CCR2对复发性肿瘤的治疗效果，为改善复发性髓母细胞治疗现状提供思路。

髓母细胞瘤是儿童颅内最常见的恶性肿瘤，大部分患者存在原位复发或沿脑脊液播散转移，而复发后五年生存率尚不足10%，对于复发髓母细胞瘤的治疗目前仍存在巨大挑战。髓母细胞瘤干细胞（medulloblastoma stem-like cells, MBSCs）表现为无限增殖、自我更新并抵抗放化疗等特征，是肿瘤复发的主要根源。但MBSCs仅靠自身并不足以保持不断更新并促进复发，必须与肿瘤微环境中基质成分如星形胶质细胞等并相互依存。因此，本项研究探索复发髓母细胞瘤中MBSCs与肿瘤相关星形胶质细胞（tumor-associated astrocyte, TAA）相互关系，并力图通过靶向肿瘤微环境改善对复发肿瘤的治疗现状。.本研究以复发髓母细胞瘤为研究对象，首先就纳入的髓母细胞瘤病例整理分析其临床病理特征及预后随访信息，筛选复发肿瘤患者；通过免疫荧光等手段检测MBSCs及TAA相关标记物在原发及复发肿瘤中的表达，并明确其与预后相关性；分离MBSCs及TAA后，通过体内或体外实验调控CCL2基因表达及培养上清中抗体封闭等手段探究其对肿瘤干性及增殖的特征的影响；通过改变CCL2影响的外界环境后检测MBSCs中Notch信号通路的活化情况进一步说明其调控肿瘤干性的分子机理。.我们发现在复发髓母细胞瘤中MBSCs比例明显升高，TAA因对放化疗不敏感，被进一步富集活化并释放CCL2；分析肿瘤组织CCL2表达与Nestin及Ki-67表达水平呈正相关，生存分析显示CCL2高表达患者预后不佳，进一步说明了CCL2表达对肿瘤恶性行为起到促进作用。在通过ELISA明确了CCL2主要来源于TAA后，将MBSCs及TAA通过细胞共培养及共同移植至小鼠体内发现TAA对MBSCs的显著滋养促进增殖功能；在MBSCs中干扰CCL2/CCR2可观察到下游Notch信号通路活化显著收到抑制。在颅内转移移植瘤模型中靶向CCL2能够显著抑制肿瘤增长。本研究从肿瘤微环境角度阐明了髓母细胞瘤复发播散的机理，为新型靶向治疗策略研发提供参考依据。