骨肉瘤细胞来源外泌体诱导CCL22分泌招募gMDSC塑造肺转移前龛的作用及机制研究

Pre-metastatic niche is a key influencing factor in the formation of tumor lung metastasis. We found that granulocytic myeloid-derived suppressor cells (gMDSCs) were profoundly increased in the lung pre-metastatic niche of osteosarcoma. Depleting of gMDSCs using anti-ly6g antibody significantly inhibited lung metastasis. Further study indicated that osteosarcoma cells derived exosomes facilitate the formation of pre-metastatic niche in the lung. Moreover, primary osteosarcoma cells induced CCL22 secretion in the pre-metastatic lung, which could recruit gMDSC infiltration. Hence, we put forward the hypothesis that osteosarcoma cell-derived exosomes act on pulmonary stromal cells, recruiting gMDSCs through CCL22-CCR4 chemokine-chemokine receptor network. This project will identify the interaction mechanism among osteosarcoma cells, pulmonary stromal cells and gMDSCs in transgenic mice and various murine osteosarcoma models. Single-cell RNA-seq will be adopted to reveals the heterogeneity of gMDSCs and specific markers for subgroup typing. Furthermore it will be promising to assess the feasibility of SPECT imaging with 99mTc-FATP2 antibody, a published gMDSCs specific marker, to detect gMDSCs and identify the progression of disease. The successful implementation of this project will establish the distribution map of gMDSCs subtypes in the pre-metastatic niche of osteosarcoma, and illustrate the feasibility of visualizing the pre-metastatic niche using FATP2-antibody based SPECT imaging.

肺转移前龛（pre-metastatic niche）是肿瘤肺转移形成的关键影响因素。前期发现骨肉瘤肺转移前龛中富集粒细胞型髓源性抑制细胞（gMDSC），靶向清除gMDSC可抑制肺转移；深入研究发现骨肉瘤外泌体介导CCL22分泌间接招募gMDSC浸润。由此我们推测“骨肉瘤细胞来源外泌体作用于肺间质细胞，诱导其分泌CCL22，通过CCL22-CCR4招募gMDSC形成肺转移前龛，协助肿瘤肺转移”。本项目将在转基因小鼠、不同骨肉瘤细胞移植瘤模型中，明确骨肉瘤细胞、肺间质细胞及gMDSC之间沟通互作机制；通过单细胞RNA-seq，分析肺转移前龛中gMDSC异质性，揭示其内在亚群分型标记；进一步建立由同位素标记FATP2抗体特异性靶向标记gMDSC，通过SPECT检测骨肉瘤肺转移前龛的方法。本项目成功开展将建立骨肉瘤肺转移前龛gMDSC亚型分布图谱，并为靶向gMDSC早期可视化及治疗肺转移提供依据

骨肉瘤是儿童与青少年最常见的原发性恶性骨肿瘤，极易发生肺转移。近十年来，骨肉瘤尤其是肺转移的治疗进入一个瓶颈期，无法取得明显的进展，单纯靶向骨肉瘤细胞本身可能难以取得较好治疗效果。因此，需要从骨肉瘤肺转移的关键影响因素——肺转移前龛出发，寻找突破目前骨肉瘤肺转移治疗瓶颈的策略。本项目通过全面分析骨肉瘤肺转移前龛免疫微环境的构成，进一步明确肺转移前龛中重要组分gMDSC的作用及机理，揭示骨肉瘤细胞通过细胞外囊泡这一重要沟通介质，实现肿瘤细胞与肺间质巨噬细胞相互沟通，诱导巨噬细胞分泌CXCL2招募gMDSC，从而塑造免疫抑制肺转移前龛，促进肺转移形成的过程。为更全面认识骨肉瘤肺转移及开发靶向该过程关键细胞及分子的治疗及监测策略提供理论依据。进一步，通过分析骨肉瘤免疫微环境显著浸润的巨噬细胞，我们确定以CD47这一热点巨噬细胞免疫检查点作为靶点，开发了具有近红外成像、免疫治疗和声动力治疗的多功能一体化纳米药物MPIRx，体内外实验结果充分显示该纳米药物具有较好安全性，同时能够显著激活机体免疫反应，从而抑制骨肉瘤生长及肺转移形成。首次验证了CD47免疫检查点治疗和声动力治疗联合应用于骨肉瘤的治疗潜能。该新型免疫治疗纳米药物具有较好的临床转化应用前景。后续可进一步优化药物设计及制作工艺，并探索开展临床试验。可为骨肉瘤患者尤其是肺转移患者提供新的有效免疫治疗策略，从而提高患者生存预后。