胶质母细胞瘤通过EGFR/c-Src/CD47信号轴抑制肿瘤相关巨噬细胞的吞噬作用

Glioblastoma is a common primary malignant brain tumor, which has rapid progression, terrible prognosis and poor response to clinical treatment. Therefore, it is imperative to find new therapeutic targets and explore new therapeutic regimens. Our previous study showed that numerous of macrophages were infiltrated in GBM specimens, while less T lymphocytes were found in the specimens. Moreover, CD47 abundance was positively correlated with malignant grade of gliomas, and the protein level of CD47 was regulated by EGFR/c-Src signal. Accordingly, we speculate that activation of EGFR promotes the escape of GBM cells from macrophage attack by up-regulating CD47. Based on the mutation and microenvironment of GBM, we will explore the feasibility of CD47 as a target for precise immunotherapy of GBM, clarify the specific mechanism of EGFR/c-Src regulating CD47 expression, and finally demonstrate whether the combination of EGFR inhibitor Afatinib and CD47 antibodies can more effectively strengthen the phagocytosis of macrophage and significantly improve the therapeutic efficacy of GBM. Researches mentioned above will be carried out through in vitro studies, animal experiments and clinical data analysis. Taken together, this study will enrich the understanding of GBM immune escape and provide a new method for GBM immunotherapy.

胶质母细胞瘤（glioblastoma，GBM）是常见的原发性恶性脑肿瘤，其特点是病程发展快、预后差和临床治疗困难。因此，寻找新的治疗靶点，探索新的治疗方案势在必行。申请者在前期研究中发现在GBM标本中浸润了大量的巨噬细胞，而T淋巴细胞较少；CD47丰度与胶质瘤恶性分级呈正相关，并且CD47蛋白水平受EGFR/c-Src信号调节。综上推测EGFR信号通过上调CD47促进GBM细胞逃离巨噬细胞的攻击。本课题从GBM的突变特点及所处的微环境角度出发，通过体外实验、动物实验和临床资料分析，探索CD47作为GBM精准免疫治疗靶点的可行性，并明确EGFR/c-Src调控CD47表达的具体机制，论证EGFR靶向药Afatinib与CD47抗体联用能否更有效地促进巨噬细胞的吞噬作用从而提高GBM的治疗疗效。本研究将丰富人们对GBM免疫逃逸的认识，并为GBM免疫治疗提供新思路。

肿瘤细胞通常通过高表达免疫检查点蛋白，如CD47等来促进肿瘤免疫逃逸。但是关于在肿瘤发生发展过程中扮演重要角色的受体酪氨酸激酶激活异常是否影响和如何调控CD47尚未可知。在本研究中，我们证明了EGFR激活通过非转录翻译水平调控CD47的表达。EGFR激活后能促进肿瘤中c-Src与CD47的结合，引起由c-Src介导的CD47的288位酪氨酸磷酸化。CD47 Y288位的磷酸化有效的抑制E3泛素连接酶Trim21与CD47的相互作用，从而阻断Trim21介导 CD47的99位和102位赖氨酸多泛素化和CD47的降解。基于原位同种小鼠脑胶质瘤模型，我们明确了肿瘤细胞中CD47 Y288F能有效的降低CD47的表达，促进巨噬细胞对肿瘤细胞的吞噬，有效的抑制肿瘤的生长和延长模型动物的生存期；然而CD47 K99/102R则显著提高肿瘤CD47的表达水平，从而抑制巨噬细胞的吞噬作用，造成肿瘤的快速进展和小鼠的死亡。更为有趣的是，我们通过CD47抗体阻断CD47/SIRPa信号轴，联合靶向EGFR治疗，相较于单药治疗能更为有效的抑制小鼠胶质瘤进展。另外，在人胶质瘤标本中CD47表达水平，与EGFR pY1068和Src pY416的水平呈正相关。本研究阐明了在EGFR激活异常的肿瘤细胞中CD47上调的分子机制，同时明确了EGFR/c-Src/Trim21/CD47信号在肿瘤免疫逃逸中的作用，论证了在肿瘤治疗中联合阻断EGFR和CD47新策略的理论可行性。