Role of tumor-associated macrophage exosomes in regulating proliferation, migration and invasion of laryngeal carcinoma cells
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摘要:背景
肿瘤相关巨噬细胞外泌体通过传递信号分子调控肿瘤细胞的生物学行为，但在喉癌的研究中鲜有报道。
目的探讨肿瘤相关巨噬细胞外泌体对喉癌细胞增殖、迁移、侵袭能力的影响。
方法使用生物信息学方法分析头颈鳞癌与肿瘤相关巨噬细胞的关系；诱导并鉴定M0和M2型肿瘤相关巨噬细胞，采用RT-qPCR、Western blot实验对其表达的细胞表面标志物IL-10、Arginase-1 (Arg-1)、CCL-18、CD206、CD-163进行检测；提取并鉴定M0和M2型巨噬细胞分泌的外泌体(M0-exo和M2-exo)，采用透射电镜法观察外泌体特有结构，纳米颗粒追踪法测定外泌体粒径，Western blot实验检测其表达的标志性蛋白HSP70；将人喉癌细胞(AMC-HN-8)分为M0-exo组和M2-exo组，其中M0-exo组为实验组，M2-exo组为对照组。采用CCK-8实验和细胞克隆形成实验检验M0-exo和M2-exo对细胞增殖能力的影响，细胞划痕实验检验M0-exo和M2-exo对单层细胞运动能力的影响，Transwell实验检验M0-exo和M2-exo在3D环境中对细胞运动能力的影响，Western blot实验检测N-钙黏蛋白、波形蛋白、E-钙黏蛋白以验证M2-exo是否对喉癌细胞的上皮间质转化进程产生影响，进而影响细胞的迁移、侵袭能力。
结果根据生物信息学分析，发现头颈鳞癌中巨噬细胞富集情况明显；分析其中免疫细胞的组成情况，发现M2型巨噬细胞占13%；培养成功的M2型巨噬细胞表达其特有的细胞表面标志物。M2-exo符合外泌体的结构和生物学特征。与M0-exo组相比，M2-exo组AMC-HN-8细胞的增殖、迁移、侵袭能力增强，差异均有统计学意义(P＜0.05)。
结论M2型肿瘤相关巨噬细胞外泌体可促进喉癌细胞的增殖、迁移和侵袭能力。
Abstract:BackgroundTumor-associated macrophage (TAM) exosomes regulate the biological behavior of tumor cells by transferring certain signaling molecules. These biovesicles have been extensively studied in breast cancer and colorectal cancer, but there is limited research on their role in laryngeal cancer.
ObjectiveTo investigate the effects of TAM exosomes on the proliferation, migration, and invasion capacity of laryngeal cancer cells.
MethodsBioinformatics analysis was employed to explore the relationship between head and neck squamous cell carcinoma and TAMs, M0 and M2 TAMs were induced and characterized using RT-qPCR and Western blotting. The exosomes secreted by M0 and M2 macrophages (M0-exo and M2-exo) were extracted and characterized using transmission electron microscopy, nanoparticle tracking, and Western blotting. Human laryngeal cancer cells (AMC-HN-8) were divided into M0-exo and M2-exo groups, and the effects on cell proliferation were assessed using the CCK-8 assay and cell cloning formation assay. The impact on the motility of monolayer cells was evaluated using the cell scratch assay, while the 3D cell motility was assessed using the Transwell assay. The effect of M2-exo on the epithelial-mesenchymal transition (EMT) process of laryngeal cancer cells, and consequently on their migration and invasion capacity, was validated by Western blotting.
ResultsBioinformatics analysis revealed a significant enrichment of macrophages in head and neck squamous cell carcinoma. The composition of immune cells within the tumor showed a higher expression proportion of M2 TAMs. Cultured M2 TAMs expressed characteristic cell surface markers. M2-exo exhibited the structural and biological features of exosomes. Compared to the M0-exo group, the M2-exo group showed significantly increased proliferation, migration, and invasion capacities of AMC-HN-8 cells, with statistically significant data.
ConclusionM2 TAM exosomes can promote the proliferation, migration, and invasion capacities of laryngeal cancer cells.
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图 2 头颈鳞癌中免疫细胞的组成情况
Figure 2. Composition of immune cells in head and neck squamous cell carcinoma
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图 3 M2型巨噬细胞的鉴定
A：RT-qPCR检测M2巨噬细胞表面标志物；B：Western blot检测IL-10、Arg-1蛋白表达。
Figure 3. Identification of M2 macrophages
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图 4 肿瘤相关巨噬细胞外泌体鉴定(电镜扫描，5 000 × )
A：透射电子显微镜观察结果；B：TAMs-exo的粒径分布图；C：HSP70标志蛋白的表达。
Figure 4. Identification of tumor-associated macrophage exosomes (SEM, 5 000 × )
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图 5 肿瘤相关巨噬细胞外泌体对喉癌细胞增殖能力的影响
A：克隆形成实验；B：克隆形成实验定量分析；C：CCK-8实验验证M2-exo对AMC-HN-8细胞增殖能力的影响。
Figure 5. Effect of tumor-associated macrophage exosomes on proliferation of laryngeal carcinoma cells
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图 6 肿瘤相关巨噬细胞外泌体对喉癌细胞迁移、侵袭能力的影响
A：划痕实验；B：划痕实验定量分析；C：Transwell细胞迁移侵袭实验；D：Transwell实验定量分析。
Figure 6. Effect of tumor-associated macrophage exosomes on migration and invasion of laryngeal carcinoma cells
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图 7 实验检测E-钙黏蛋白、N-钙黏蛋白、波形蛋白的表达水平
A：Western blot实验；B：Western blot实验定量分析。
Figure 7. The expression levels of E-cadherin，N-cadherin and vimentin were detected
表 1 引物序列
Table 1 Primer Sequence
目的基因 引物序列(5’-3’) GAPDH F：CCATGGGGAAGGTGAAGGTC
R：GAAGGGGTCATTGATGGCAACIL-10 F：TCTCCGAGATGCCTTCAGCAGA
R：TCAGACAAGGCTTGGCAACCCACCL-18 F：GTTGACTATTCTGAAACCAGCCC
R：GTCGCTGATGTATTTCTGGACCCCD206 F：AGCCAACACCAGCTCCTCAAGA
R：CAAAACGCTCGCGCATTGTCCACD163 F：CCAGAAGGAACTTGTAGCCACAG
R：CAGGCACCAAGCGTTTTGAGCT
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