超声触发载GW4869微泡干预肿瘤外泌体以防控肝癌射频消融后复发的效果与机理研究

Hepatocellular carcinoma (HCC) is a common malignant tumor with high mortality. Radiofrequency ablation (RFA) can effectively treat HCC, but some patients may suffer recurrence. To prevent cancer recurrence and metastasis is important for improving long term therapeutic effect of RFA for HCC. The mechanism of HCC recurrence after RFA has not been elucidated completely. Current studies show that functions of exosomes involve in normal physiology of cells, dissemination of oncogenes from tumor cells, carcinogenesis, tumor metastasis and recurrence, constitution of tumor microenvironment, etc. We hypothesized that exosomes play a vital role in the HCC recurrence after RFA. If exosomes of primary HCC load carcinogenic constituents to the tumor microenvironment, peripheral liver cell and other cells, then it will increase the probability to induce generation of new foci of HCC. On this condition, if synthesis and secretion of exosomes are inhibited and contained before and during the process of RFA for HCC, the recurrence and metastasis of HCC may be avoided or reduced. The goals of this project are to formulate a kind of ultrasound response nanobubbles loading with an exosome inhibitor of GW4869 and crack them in the HCC to release the inhibitor to inhibit the synthesis and secretion of exosomes, so that to avoid or reduce recurrence of HCC after RFA, to improve the therapeutic effect and to explore relevant mechanisms. The main contents of this project include elevated temperature on the influence of synthesis and secretion of exosomes of liver cancer cells, liver cells, tumor associated fibroblast (TAF), and tumor associated macrophage (TAM); extraction and isolation of exosomes of these cells in vivo and vitro; identification of miRNAs in the exosomes of those cells; analysis of the substance (miRNA, HIF-1 α, etc) transferring function of exosomes between different cells, and the expression and regulation of miRNA-122*, HIF-1 α, and Mdm2; changing and transferring of exosomes of tumor after RFA; histopatholigical analysis of HCC after RFA of animal; assessment of GW4869 on the inhibiting effect of recurrence and metastasis after RFA; formulation and characterization of nanobubbles loading with GW4869 using PLGA and C3F8 gas by double emulsifying protocol; the releasing rate of GW4869 while the nanobubbles are cracked by ultrasound, etc, the effect of GW4869 on the synthesis and secretion of exosomes of HCC in vitro and vivo. The significance of this project is to further determine the roles of exosomes in the tumor metastasis and recurrence, to probe the mechanisms of HCC recurrence after RFA, and to find a potential new method for translational medicine of HCC therapy.

射频消融治疗肝癌的近期疗效好，但部分患者会复发。复发的确切机理还未阐明。基于外泌体有多种生物学功能，与正常生理、多种疾病和肿瘤发生及转移有关的前期研究，我们假设：射频消融治疗肝癌后复发与外泌体有关，干预肿瘤外泌体能防控癌症复发和转移。为验证假设，我们拟用能抑制细胞外泌体合成和释放的GW4869进行干预研究，探讨效果和有关机理。但GW4869对癌细胞和正常细胞都起作用，静脉给药应用时将有明显副作用，为此我们拟制备新型制剂克服，用超声波在肝癌瘤体触发载GW4869微泡破裂，定点集中释放GW4869，以抑制外泌体，在控制副作用前提下减低或防止射频消融后复发。项目由体内外实验组成：温度增加对细胞外泌体的影响；外泌体成分分析、成分交换及机制；射频消融后瘤体外泌体的变化；复发和转移实验；双乳化法制备载GW4869纳米微泡及相关评估。项目成功完成将为防止肝癌射频消融后复发提供新思路，为转化医学奠定基础。

肝细胞癌射频消融治疗后可发生转移，原理被推测认为与治疗期间外泌体将癌细胞内miRNA等活性物质向外转运后影响其他细胞有关；干预癌细胞外泌体合成和释放有可能减少或抑制转移。主要研究内容：提取肝癌细胞等细胞外泌体并进行定征和鉴定；检测肝癌细胞外泌体与瘤体内其它细胞（巨噬细胞）外泌体间（外泌体与肿瘤微环境研究）有无成分交换及相关变化；用GW4869干预细胞外泌体合成和释放；制备载 GW4869 微泡并进行定征、生物相容性评估、载药率及释药率分析、对肝癌细胞迁移和侵袭能力影响；用GW4869干预荷瘤裸鼠探讨能否抑制肿瘤生长或转移。主要结果：肝癌细胞与巨噬细胞共培养后，巨噬细胞形态由规整变为不规则；与肝癌细胞外泌体共孵育后的巨噬细胞的M2型标记物CD206以及Arg-1的mRNA的表达显著高于正常肝细胞外泌体组；与肝癌细胞外泌体共孵育后的巨噬细胞的M2型标记物IL-10、TGF-β的蛋白表达水平显著高于正常肝细胞外泌体组；肝癌细胞外泌体能够进入巨噬细胞；与M2极化的巨噬细胞外泌体共孵育后的肝癌细胞迁移及侵袭能力显著增强；在模拟肝癌细胞与巨噬细胞的肿瘤微环境中加入超声辐照的载GW4869微泡后，肝癌细胞的迁移及侵袭能力较对照组显著下降；GW4869干预荷瘤裸鼠未能使得瘤体变小或消失；索拉非尼和索拉非尼联合GW4869干预荷瘤鼠不能使得瘤体变小或消失，但瘤体的生长趋势得到抑制。结论：肝细胞癌外泌体的miR-151可诱导瘤体中的巨噬细胞极化为肿瘤相关巨噬细胞，肿瘤相关巨噬细胞对肝癌细胞迁移及侵袭有促进作用；超声辐照的载GW4869微泡能够通过抑制肝癌细胞与微环境细胞间的外泌体通讯而降低肝癌细胞的迁移及侵袭能力；荷瘤鼠动物实验中，GW4869干预荷瘤裸鼠未能使得瘤体变小或消失。科学意义：肝细胞癌细胞来源外泌体成分对肝癌细胞迁移及侵袭有促进作用，通过GW4869抑制癌细胞与微环境细胞间的外泌体通讯有可能抑制癌症转移，这对完整阐明肝癌转移机制和干预治疗有意义。