外泌体-TM4SF3多阶段调控MSCs分化促进乳腺癌骨转移的机制研究

Bone metastases multi-stage process is composed of EMT dynamic regulation, abnormal angiogenesis, andmicroenvironment transformation. Our previous study found that the four transmembrane superfamily protein 3 (TM4SF3) play an important role in this process. Exosomes (Exos) - TM4SF3 induced EMT and enhanced the proportion of cancer stem cells, thus promoting tumor invasion, metastasis and treatment resistance. The expression of TM4SF3 in bone marrow Exos and mesenchymal stem cells (MSCs) of patients with bone metastasis was significantly increased, and MSCs were differentiated into osteoblasts and abnormal angiogenesis by reason of TM4SF3. Based on previous studies, the project will focus on "hypoxia / EGF-TM4SF3 increased -- abnormal angiogenesis (first stage of regulation) -- Exos-TM4SF3high distant signaling to regulate MSCs differentiation (second stage of regulation) -- cancer cells colonization". Molecular biology means will be conducted to elucidate the effect of Exo-TM4SF3high on the differentiation of MSCs into vascular endothelial cells and osteoblasts, and clarify the molecular chaperone and core signaling pathway of TM4SF3 interaction. We will also study the effects of Exo-TM4SF3high on other related events in MSCs differentiation regulation like bone synthesis / catabolic changes, T cells immune escape and so on. To explore the effect of Exo-TM4SF3high on osteoclast function, bone microvascular formation and tumor cell recruitment by animal model and in vivo imaging will be tested. The clinical data of Exo-TM4SF3high as a biomarker of breast cancer bone metastasis (blood, urine ) and its potential clinical application will be explored.

EMT 动态调控、异常血管生成、微环境重塑组成骨转移的多阶段过程，我们前期研究发现四次跨膜超家族蛋白3（TM4SF3）在其中发挥重要作用：乳腺癌骨转移患者血清/骨髓外泌体及间充质干细胞（MSCs）TM4SF3 表达显著增高，对MSCs 向成骨细胞分化及异常血管生成具调控作用。在前期基础上，本项目将围绕“乏氧/EGF——TM4SF3 作用于肿瘤周围MSCs 与血管形成(第一阶段)——外泌体TM4SF3 远处信号传递，调控骨髓MSCs 成骨细胞分化（第二阶段）”为主线，采用分子生物学手段阐明外泌体TM4SF3 对MSCs 多阶段分化调控的影响，明确分子伴侣及核心通路；探讨MSCs 分化调控中其他伴随相关事件如骨代谢性因子、肿瘤细胞招募等变化；利用动物模型及体内成像检测外泌体TM4SF3 对破骨细胞功能、骨微血管形成的影响；扩大临床样本验证，探讨作为乳腺癌骨转移的生物预测标记物及潜在临床应用。

乳腺癌远处转移是导致患者死亡和治疗失败的主要原因。转移并非随机过程，是包括乳腺癌细胞自身干性增强与肿瘤微环境重塑的多阶段复杂反应过程，使利于乳腺癌细胞从原发灶的脱落、逃逸和远处脏器的定植。既往研究揭示：肿瘤细胞分泌的外泌体在转移信号传递中起重要作用，但对其机制尚不完全了解。相比较其他分子亚型，三阴性乳腺癌（TNBC）具有增强的干性特征，临床上表现为容易肺转移，但骨转移发生率较低。造成这一现象的分子机制是什么目前并不清楚。本项目聚焦这一重要科学问题，按照预定研究计划，围绕干性增强的四次跨膜超家族蛋白3（TM4SF3，又称TSPAN8）阳性的乳腺癌细胞容易远处转移，系统性揭示了TSPAN8+乳腺癌细胞分泌富集TSPAN8的外泌体（TEVs-TShigh）、通过TEVs-TShigh信号传递进行转移前微环境改造、主导转移器官选择性的机制：①阐明了TEVs-TShigh对乳腺癌原发灶及远处脏器微环境中的关键亚群细胞尤其是骨髓来源间充质干细胞（Mesenchymal Stem Cells, BM-MSCs）的调控，促进其向成骨细胞的分化从而抑制骨转移的发生；②揭示了TEVs-TShigh通过对免疫微环境中的抑制性调节T细胞亚群调控促进免疫逃逸与肺转移的分子机制，研究了TSPAN8相互作用的分子伴侣及关键调控信号通路；③在这些系列性研究基础上，研究者采用噬菌体库筛选，研发了靶向TSPAN8的特异性单克隆治疗性抗体，体内外疗效评估显示TSPAN8特异性抗体治疗可有效阻断TNBC细胞远处转移的发生，显示良好的临床应用前景。