血小板参与的瘤栓形成在骨肉瘤肺转移中相关机制研究

Osteosarcoma is the most frequent malignant primary tumor of bone in children and adolescents with an approximately 5-year survival about 50-60%. Lung metastasis remains as the most challenging condition during treatment and follow-up, which has been regarded as the main reason for the bottleneck of prognosis. Tumor thrombus is occasionally diagnosed in patients with malignant tumor. It usually exists during tumor angiogenesis associated with proliferation and metastasis. The presentation shows aggressive features and advanced stage of the disease. Tumor thrombus is associated with high metastasis potential. Based on the evolution theory of tumor metastasis, tumor thrombus can be the intermediate phase between local invasion and distal metastasis. In order to investigate the underlying mechanism and identify candidate metastasis driver genes, we collected a set of high quality paired tumor tissue, both the primary site and corresponding tumor thrombus that were treated by thrombectomy. Unbiased high throughput approaches for identifying expression changes between tumor and normal samples can reveal significant changed markers and pathways. By RNA-seq, we explored the transcriptomic signatures of osteosarcoma patients with tumor thrombus. Based on the preliminary results from 3 patients, the dysregulated gene between the primary site and tumor thrombus suggested that activation of complement and coagulation cascades was of significance. Activation of coagulation cascades induced platelets activation. Meanwhile, 5-hmc levels of circulation DNA in patients with osteosarcoma showed significantly increased compared with healthy people. Integrated pathway analysis also revealed platelet activation. Taking together, the initial results from two projects both showed significant role of platelet in tumor thrombus formation and distal metastasis. The objective of the current project is to explore the mechanism of platelet-induced tumor thrombus formation and lung metastasis by in vitro, animal model, and clinical samples. The findings may also contribute to the development of novel therapeutic approaches and molecular evidence.

骨肉瘤是儿童及青少年最常见的恶性骨肿瘤，5年存活率仅为50-60%，肺转移是目前治疗难点。瘤栓是恶性肿瘤伴随的一种临床现象，是肿瘤增殖转移过程中侵犯血管的表现，与转移高度相关，合并瘤栓的患者较早出现转移且预后很差。前期通过比较肿瘤原发灶与配对瘤栓转录组差异，我们发现瘤栓形成与凝集素通路异常高度相关，后者引起血小板活化。此外，通过对比骨肉瘤患者与正常人群外周血DNA的5-羟甲基胞嘧啶（5-hmC）水平发现，骨肉瘤患者5-hmC含量高于正常人群，且相关基因与血小板活化高度相关。以上结果均提示血小板在骨肉瘤瘤栓形成及转移方面有着十分重要的作用。本项目拟针对骨肉瘤中血小板参与的瘤栓形成在肺转移过程中的相关机制进行研究，利用细胞实验、动物模型、临床标本，明确血小板在骨肉瘤增殖、瘤栓形成及远处转移方面的作用，并阐明相关分子机制，为治疗骨肉瘤转移提供新的思路及重要理论依据。

骨肉瘤是儿童及青少年最常见的骨恶性肿瘤，发病率占全部恶性骨肿瘤的27.5%。骨肉瘤恶性程度高，发病隐蔽，就诊时80%以上的患者存在肺转移，这些特点给治疗带来很大的困难。近年来，随着诊治技术的发展，骨肉瘤患者的5年存活率已达到58-76%，但该治疗效果在过去的二十年中没能进一步提高，治疗一直处于瓶颈期，这主要是由于30-40%的患者在诊断或治疗过程中出现肺转移、骨转移等远处转移，且现有治疗手段无法有效控制转移灶进展，最终由于肿瘤进展导致死亡。合并瘤栓骨肉瘤患者往往出于进展期，有极高的复发率和转移风险，整体预后差，本项目通过极端临床现象入手，通过瘤栓作为研究切入点，初步证实了这一现象产生的主要始动因素是血小板参与的，通过分泌型细胞因子调控骨肉瘤细胞的增殖及转移能力，我们发现了补体凝集通路异常，但在研究总我们发现可能存在F3-TGF-β的正反馈循环，但该循环的具体调控机制尚不明确，并且瘤栓转移过程汇中是依赖血小板FAK的，通过转基因鼠，我们特定敲除了小鼠血小板的FAK表达，进而我们发现肿瘤转移能力显著下降，并且瘤栓形成能力大大降低。通过本项目研究我们提出肿瘤在远处形成转移灶能力可能是非出血管依赖性的即 non-extravasation dependent metastasis，也就是是说当肿瘤细胞在肺的毛细血管滞留时，由于其与血小板形成的微小瘤栓复合体可以与肺血管内皮细胞粘附，进而通过内皮细胞的作用刺激血小板释放TGF-β直接刺激骨肉瘤细胞增殖，在管腔内形成转移灶，而不需要按原有理论穿出血管壁在进一步增殖形成转移灶。