椎体红骨髓分泌的CX3CL1调控Src/FAK通路对脊柱转移性肿瘤的促进作用及分子机制

Skeletal metastases are found in 60% of cancer patients, and the spine accounts for 50% of these osseous metastases, leading to paraplegia and urinary and fecal incontinence. But the underlying reasons for the high incidence of spinal metastases(SM) remain unclear. Recently, several studies have shown that abundant red bone marrow in the vertebral body is a key factor related to the higher incidence of SM, compared to that of limb metastasis. The microenvironment of red bone marrow contains many types of cytokines, including chemokines. However, no researches evaluating the role of chemokines in SM have been reported. In our preliminary work, we used the gene chip technique to screen a amount of tissue samples. Compared to normal spinal bone samples and primary spinal tumor samples, CX3CL1, an important chemokine, was up-regulated significantly in the bone samples with SM, and our in vitro experiment showed antibodies of CX3CL1 can significantly inhibited the migratory ability of tumor cells. High throughput analysis showed that Src/FAK pathway was the major downstream signaling pathway activated by CX3CL1. Therefore we speculate that the high expression of CX3CL1 in vertebral bone marrow may promote SM by binding to CX3CR1-bearing tumor cells, which is followed by the activation of Src/FAK pathway. Based on our previous findings, we apply this grant to figure out the role and molecular mechanisms of CX3CL1/CX3CR1 in SM. Then we will separate circulating tumor cells (CTC) to verify the role of CX3CL1 in SM. Consequently, the molecular mechanism of SM may be clarified in terms of signaling pathway of CX3CL1/CX3CR1 by our research, which may form a theoretical foundation for the utility of CX3CL1 as an indicator for early SM detection.

恶性肿瘤容易发生骨转移尤其是脊柱骨转移，会引起截瘫、大小便失禁等严重后果。但脊柱转移性肿瘤（SM）高发原因仍然不明。研究发现椎体丰富的红骨髓是脊柱转移发生率高于四肢骨的关键。红骨髓微环境中趋化因子是否对肿瘤细胞迁移到脊柱起作用还未见报道。我们前期研究利用转移瘤椎体的表达谱芯片，筛查发现趋化因子CX3CL1比正常椎体和原发肿瘤椎体表达明显升高，其抗体能够显著抑制肿瘤细胞的体外迁移，高通量分析表明Src/FAK通路可能是CX3CL1潜在的作用靶点，提示红骨髓中高表达的CX3CL1与受体CX3CR1结合后可能通过上调Src/FAK通路促进肿瘤细胞转移到脊柱。为证明该假说，本项目旨在明确CX3CL1促进肿瘤细胞转移到脊柱的作用及分子机制，并利用循环肿瘤细胞（CTC）分离来验证CX3CL1在CTC转移到脊柱中的作用。本项目有望揭示CX3CL1促进脊柱转移的分子机制，为早期发现及预防SM提供新靶点。

本项目拟阐明 CX3CL1 对肿瘤细胞的调节作用及其对脊柱转移性肿瘤（Spinal Metastasis，SM）的影响，从新的视角解释脊柱好发转移性肿瘤的原因，对脊柱转移性肿瘤的早期发现和预防提供新的理论依据，为将来的防治策略提供新的靶点。首先通过基因表达谱芯片技术、免疫组化法等探讨多种原发肿瘤出现脊柱转移时，肿瘤旁松质骨微环境的基因表达、趋化因子表达情况。其次采用流式细胞术、CCK-8法、划痕实验、Transwell小室法等观察并分析肺癌和乳腺癌细胞的增殖、黏附、侵袭及迁移能力。使用分子生物学手段干预信号通路上的关键分子，进一步探索CX3CL1与CX3CR1结合后，肿瘤细胞内的信号转导通路，揭示CX3CL1促进肿瘤转移至脊柱的具体机制。最后建立脊柱转移瘤动物模型，术后8周通过影像学等方法进一步在体内验证CX3CL1在脊柱转移瘤中的作用。研究发现1、CX3CL1可能是参与多种原发肿瘤发生脊柱转移的关键趋化因子，其可成为肿瘤患者预后转归的预测因素和潜在的治疗靶点，并且CX3CL1在椎体松质骨中表达量较四肢松质骨高；2、CX3CL1/CX3CR1通过激活Src/FAK信号通路促进肺癌、乳腺癌细胞的迁移及侵袭，而信号通路阻断剂Saracatinib、Bosutinib等可以阻断这一过程；3、在动物实验中，注射过表达CX3CR1的乳腺癌细胞成瘤率高于注射普通乳腺癌细胞，进一步说明了CX3CL1的促乳腺癌脊柱转移的作用。本项目课题基本完成了预定的研究目标，并且已发表论文8篇，科普文章4篇。