MDSCs介导的骨肉瘤对PD-1抗体治疗不敏感的机制研究

Anti-PD-1 therapy is a completely new method for cancer treatment. Initial clinical studies of antibodies directed against PD-1 showed both an encouraging safety profile and remarkable antitumor activity in subsets of patients with melanoma and lung cancer. However, patients with osteosarcoma showed poor response to anti-PD-1 therapy, the mechanism of which is not well understood. In our preliminary experiment, we found that the curative effect of anti-PD-1 therapy is dependent on the widespread infiltration of CTL into the tumor tissue. Further study revealed that lots of CTL-inhibiting MDSCs migrate into the osteosarcoma microenvironment along a CXCL12 concentration gradient. The binding of CXCL12 to CXCR4 results in the activation of downstream AKT pathway that mediates reduced apoptosis of MDSCs. Based on the above results, we hypothesized that osteosarcoma tissues might recruit a great number of MDSCs and promote their survival through the CXCL12-CXCR4 biological axis; MDSCs could inhibit CTL infiltration and proliferation, which leads to poor response to anti-PD-1 therapy in osteosarcoma. On this basis, we aim to delve deeper into the MDSCs-mediated mechanism by which osteosarcoma patients show poor response to anti-PD-1 therapy. Moreover, we aim to uncover the molecular mechanisms of CXCR4 in the recruitment, survival and activation of MDSCs and to elucidate the synergistic action of CXCR4 antagonist AMD3100 and PD-1 antibody in osteosarcoma treatment. Taken together, this study could provide the basis for establishing AMD3100 and PD-1 antibody co-administration as a novel therapeutic regimen for patients with osteosarcoma. Therefore, this research project is significant to the theoretical research and practical applications.

PD-1抗体治疗是一种全新的肿瘤免疫治疗方法，但其在骨肉瘤临床应用中效果不佳，具体机制尚不清楚。我们前期实验结果显示PD-1抗体治疗依赖于CTL在肿瘤组织的广泛浸润；骨肉瘤组织中存在大量对CTL增殖有抑制作用的MDSCs；CXCL12对MDSCs有趋化作用并能活化AKT等通路来减少MDSCs凋亡。基于以上结果，提出假说：骨肉瘤组织通过CXCL12-CXCR4生物学轴募集大量MDSCs并促进其存活，继而抑制CTL生长浸润，导致PD-1抗体治疗效果不佳。本课题拟在此基础上，深入研究MDSCs介导的骨肉瘤对PD-1抗体治疗不敏感的细胞及分子机制，阐明CXCR4在MDSCs募集、存活、发挥功能过程中的作用机制，探讨CXCR4拮抗剂AMD3100在协同PD-1单抗治疗骨肉瘤中的功能意义。这些研究可为确立AMD3100联合PD-1单抗作为骨肉瘤临床治疗的全新方案提供科学依据，具有重要意义。

PD-1抗体治疗是一种全新的肿瘤免疫治疗方法，但其在骨肉瘤临床应用中效果不佳，具体机制尚不清楚。在本项目的研究中，我们采用免疫组织化学法检测了骨肉瘤组织中PD-L1、CXCR4、CD11b（髓系细胞的分子标志物）、CD8a（CTLs的分子标志物）的表达情况，并通过Kaplan-Meier生存分析法探讨了CXCR4阳性细胞及CTLs在骨肉瘤组织中浸润的临床意义。随后我们采用小鼠骨肉瘤皮下移植瘤模型及活体成像技术检测了CXCR4拮抗剂协同PD-1免疫疗法在杀伤骨肉瘤中的作用，又通过流式细胞术、激光共聚焦技术、蛋白免疫印迹技术、Transwell小室实验、T细胞增殖抑制实验等多种方法对骨肉瘤中CXCL12/CXCR4生物学轴促进MDSCs迁移、存活的分子细胞机制进行了深入研究。经过大量研究，我们发现：骨肉瘤组织中CXCR4主要表达于间质细胞并且与CTLs的浸润呈负相关；CXCR4高表达与骨肉瘤患者预后不良密切相关；同时阻断CXCR4与PD-1能显著增加小鼠骨肉瘤组织中CTLs的浸润并有效抑制骨肉瘤生长。分子细胞机制研究发现：骨肉瘤组织中存在大量CXCR4阳性的MDSCs，这些MDSCs对CTLs具有显著的抑制作用；骨肉瘤组织分泌的CXCL12具有趋化并募集CXCR4阳性MDSCs的作用，CXCL12结合MDSCs表面CXCR4受体后通过活化AKT信号通路促进MDSCs的迁移与存活，因此CXCR4拮抗剂可发挥清除骨肉瘤中MDSCs的作用。本项目揭示了MDSCs介导的骨肉瘤对PD-1抗体治疗不敏感的细胞及分子机制，阐明了CXCR4在MDSCs募集、存活、发挥功能过程中的作用机制，为确立CXCR4拮抗剂联合PD-1单抗作为骨肉瘤临床治疗的全新方案提供了充分科学依据。