前列腺癌细胞来源的外泌体诱导巨噬细胞极化
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摘要
外泌体(exosome)是直径约30～150 nm的由细胞分泌的一种具有生物学活性的囊泡。有些来自癌细胞的外泌体可以将巨噬细胞(macrophages,Mφ)极化为M2亚型,但前列腺癌细胞来源的外泌体在巨噬细胞极化中的作用仍缺乏研究。本研究采用超滤法提取前列腺癌细胞PC-3M-2B4和PC-3M-IE8条件培养基中的外泌体(PCa-exo)。分别用透射电子显微镜、纳米粒径分析及Western印迹对外泌体形态、颗粒大小和表面的特异性分子标志进行分析鉴定。用PKH67标记外泌体,观察PCa-exo能否被巨噬细胞吸收。免疫荧光分析PCa-exo处理巨噬细胞后,M2型巨噬细胞表面分子标志CD206的表达差异。用q-PCR观察PCa-exo诱导后的巨噬细胞中IL-10、IL-1β等细胞因子的表达。电镜、Western印迹和纳米粒径分析的结果显示,PCa-exo形态多为圆形,直径约为40～150 nm,PCa-exo能被巨噬细胞大量吸收。PCa-exo诱导后,巨噬细胞中CD206荧光表达显著增高,IL-10、IL-1β及IL-12等炎症因子的表达水平与M2/TAM亚型巨噬细胞的表达谱一致。本研究表明,前列腺癌细胞来源的外泌体能诱导巨噬细胞极化为M2表型。
Exosomes are biologically active vesicles secreted by cells with a diameter of 30-150 nm.Some exosomes from cancer cells can polarize macrophages( Mφ) into M2 subtypes. However,the role of prostate cancer-derived exosomes in macrophage polarization remains unknown. In this study,ultrafiltration was used to extract exosomes( PCa-exo) from conditioned medium of prostate cancer cells PC-3 M-2 B4 and PC-3 M-IE8. Transmission electron microscopy,nanoparticle size analysis and Western blotting were used to analyze and identify the shape,particle size and specific surface molecular markers of exosomes respectively. Exosomes were labeled with PKH67 to observe whether PCa-exo could beabsorbed by macrophages. The differential expression of CD206 on M2 macrophages was detected by immunofluorescence. The expression of cytokines such as IL-10 and IL-1β in macrophages induced by PCa-exo was observed by q-PCR. Our results showed that the PCa-exo morphology was mostly round and the diameter was about 40-150 nm. PCa-exo could be absorbed by macrophages in large quantities. After PCa-exo induction,the expression of CD206 in macrophages was significantly increased,and the expression levels of inflammatory factors such as IL-10,IL-1β and IL-12 were consistent with those of M2/TAM subtype macrophages. This study demonstrates that exosome from prostate cancer cells can induce macrophage polarization to the M2 phenotype.
Exosomes are biologically active vesicles secreted by cells with a diameter of 30-150 nm.Some exosomes from cancer cells can polarize macrophages( Mφ) into M2 subtypes. However,the role of prostate cancer-derived exosomes in macrophage polarization remains unknown. In this study,ultrafiltration was used to extract exosomes( PCa-exo) from conditioned medium of prostate cancer cells PC-3 M-2 B4 and PC-3 M-IE8. Transmission electron microscopy,nanoparticle size analysis and Western blotting were used to analyze and identify the shape,particle size and specific surface molecular markers of exosomes respectively. Exosomes were labeled with PKH67 to observe whether PCa-exo could beabsorbed by macrophages. The differential expression of CD206 on M2 macrophages was detected by immunofluorescence. The expression of cytokines such as IL-10 and IL-1β in macrophages induced by PCa-exo was observed by q-PCR. Our results showed that the PCa-exo morphology was mostly round and the diameter was about 40-150 nm. PCa-exo could be absorbed by macrophages in large quantities. After PCa-exo induction,the expression of CD206 in macrophages was significantly increased,and the expression levels of inflammatory factors such as IL-10,IL-1β and IL-12 were consistent with those of M2/TAM subtype macrophages. This study demonstrates that exosome from prostate cancer cells can induce macrophage polarization to the M2 phenotype.
引文
[1] Mantovani A, Sozzani S, Locati M, et al. Macrophage polarization:tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes[J]. Trends Immunol,2002,23(11):549-555
[2] Cheng Y,Zhu Y,Xu J,et al. PKN2 in colon cancer cells inhibits M2 phenotype polarization of tumor-associated macrophages via regulating DUSP6-Erk1/2 pathway[J]. Mol Cancer,2018,17(1):13
[3] Asano K, Nabeyama A, Miyake Y, et al. CD169-positive macrophages dominate antitumor immunity by crosspresenting dead cell-associated antigens[J]. Immunity,2011,34(1):85-95
[4] Sica A,Mantovani A. Macrophage plasticity and polarization:in vivo veritas[J]. J Clin Invest,122(3):787-795
[5] Sica A,Schioppa T,Mantovani A,et al. Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression:potential targets of anti-cancer therapy[J].Eur J Cancer,2006,42(6):717-727
[6] Komohara Y,Jinushi M,Takeya M. Clinical significance of macrophage heterogeneity in human malignant tumors[J]. Cancer Sci,2014,105(1):1-8
[7] Henze AT,Mazzone M. The impact of hypoxia on tumorassociated macrophages[J]. J Clin Invest,2016,126(10):3672-3679
[8] Gollapudi K,Galet C,Grogan T,et al. Association between tumor-associated macrophage infiltration,high grade prostate cancer and biochemical recurrence after radical prostatectomy[J]. Am J Cancer Res,2013,3(5):523-529
[9] Wu A,Wei J,Kong LY,et al. Glioma cancer stem cells induce immunosuppressive macrophages/microglia[J]. Neuro Oncol,2010,12(11):1113-1125
[10] Gabrusiewicz K,Li X,Wei J,et al. Glioblastoma stem cellderived exosomes induce M2 macrophages and PD-L1 expression on human monocytes[J]. Oncoimmunology, 2018, 7(4):e1412909
[11] Ham S,Lima LG,Chai EPZ,et al. Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3Signaling[J]. Front Immunol,2018,9:871
[12] Vlassov AV,Magdaleno S,Setterquist R,et al. Exosomes:current knowledge of their composition,biological functions,and diagnostic and therapeutic potentials[J]. Biochim Biophys Acta,2012,1820(7):940-948
[13] Raposo G, Stoorvogel W. Extracellular vesicles:exosomes,microvesicles,and friends[J]. J Cell Biol,2013,200(4):373-383
[14] Lakkaraju A,Rodriguez-Boulan E. Itinerant exosomes:emerging roles in cell and tissue polarity[J]. Trends Cell Biol,2008,18(5):199-209
[15] Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses[J]. Nat Rev Immunol,2009,9(8):581-593
[16] Mittelbrunn M,Sánchez-Madrid F. Intercellular communication:diverse structures for exchange of genetic information[J]. Nat Rev Mol Cell Biol,2012,13(5):328-335
[17] Nieuwland R,Sturk A. Why do cells release vesicles?[J].Thromb Res,2010,125 Suppl 1:S49-S51
[18] Mathivanan S, Ji H, Simpson RJ. Exosomes:extracellular organelles important in intercellular communication[J]. J Proteomics,2010,73(10):1907-1920
[19] Gao X,Shen X,Dong X,et al. peptide nucleic acid promotes systemic dystrophin expression and functional rescue in dystrophin-deficient mdx mice[J]. Mol Ther Nucleic Acids,2015,4:e255
[20] Qi H,Yang L,Li X,et al. Exosomes separated based on the“STOP”criteria for tumor-targeted drug delivery[J]. J Mater Chem B,2018,6:2758-2768
[21] Allavena P,Sica A,Garlanda C,et al. The Yin Yan of tumorassociated macrophages in neoplastic progression and immune surveillance[J]. Immunol Rev,2008,222:155-161
[22] Burkholder B,Huang RY,Burgess R,et al. Tumor-induced perturbations of cytokines and immune cell networks[J]. Biochim Biophys Acta,2014,1845(2):182-201
[23] Colegio OR,Chu NQ,Szabo AL,et al. Functional polarization of tumour-associated macrophages by tumour-derived lactic acid[J].Nature,2014,513(7519):559-563
[24] Wang X,Luo G,Zhang K, et al. Hypoxic Tumor-Derived Exosomal miR-301a Mediates M2 Macrophage Polarization via PTEN/PI3Kγto Promote Pancreatic Cancer Metastasis[J].Cancer Res,78(16):4586-4598
[25] Cooks T,Pateras IS,Jenkins LM,et al. Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246[J]. Nat Commun,2018,9(1):771
[26] Ying X,Wu Q,Wu X,et al. Epithelial ovarian cancer-secreted exosomal miR-222-3p induces polarization of tumor-associated macrophages[J]. Oncotarget,2016,7(28):43076-43087
[2] Cheng Y,Zhu Y,Xu J,et al. PKN2 in colon cancer cells inhibits M2 phenotype polarization of tumor-associated macrophages via regulating DUSP6-Erk1/2 pathway[J]. Mol Cancer,2018,17(1):13
[3] Asano K, Nabeyama A, Miyake Y, et al. CD169-positive macrophages dominate antitumor immunity by crosspresenting dead cell-associated antigens[J]. Immunity,2011,34(1):85-95
[4] Sica A,Mantovani A. Macrophage plasticity and polarization:in vivo veritas[J]. J Clin Invest,122(3):787-795
[5] Sica A,Schioppa T,Mantovani A,et al. Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression:potential targets of anti-cancer therapy[J].Eur J Cancer,2006,42(6):717-727
[6] Komohara Y,Jinushi M,Takeya M. Clinical significance of macrophage heterogeneity in human malignant tumors[J]. Cancer Sci,2014,105(1):1-8
[7] Henze AT,Mazzone M. The impact of hypoxia on tumorassociated macrophages[J]. J Clin Invest,2016,126(10):3672-3679
[8] Gollapudi K,Galet C,Grogan T,et al. Association between tumor-associated macrophage infiltration,high grade prostate cancer and biochemical recurrence after radical prostatectomy[J]. Am J Cancer Res,2013,3(5):523-529
[9] Wu A,Wei J,Kong LY,et al. Glioma cancer stem cells induce immunosuppressive macrophages/microglia[J]. Neuro Oncol,2010,12(11):1113-1125
[10] Gabrusiewicz K,Li X,Wei J,et al. Glioblastoma stem cellderived exosomes induce M2 macrophages and PD-L1 expression on human monocytes[J]. Oncoimmunology, 2018, 7(4):e1412909
[11] Ham S,Lima LG,Chai EPZ,et al. Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3Signaling[J]. Front Immunol,2018,9:871
[12] Vlassov AV,Magdaleno S,Setterquist R,et al. Exosomes:current knowledge of their composition,biological functions,and diagnostic and therapeutic potentials[J]. Biochim Biophys Acta,2012,1820(7):940-948
[13] Raposo G, Stoorvogel W. Extracellular vesicles:exosomes,microvesicles,and friends[J]. J Cell Biol,2013,200(4):373-383
[14] Lakkaraju A,Rodriguez-Boulan E. Itinerant exosomes:emerging roles in cell and tissue polarity[J]. Trends Cell Biol,2008,18(5):199-209
[15] Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses[J]. Nat Rev Immunol,2009,9(8):581-593
[16] Mittelbrunn M,Sánchez-Madrid F. Intercellular communication:diverse structures for exchange of genetic information[J]. Nat Rev Mol Cell Biol,2012,13(5):328-335
[17] Nieuwland R,Sturk A. Why do cells release vesicles?[J].Thromb Res,2010,125 Suppl 1:S49-S51
[18] Mathivanan S, Ji H, Simpson RJ. Exosomes:extracellular organelles important in intercellular communication[J]. J Proteomics,2010,73(10):1907-1920
[19] Gao X,Shen X,Dong X,et al. peptide nucleic acid promotes systemic dystrophin expression and functional rescue in dystrophin-deficient mdx mice[J]. Mol Ther Nucleic Acids,2015,4:e255
[20] Qi H,Yang L,Li X,et al. Exosomes separated based on the“STOP”criteria for tumor-targeted drug delivery[J]. J Mater Chem B,2018,6:2758-2768
[21] Allavena P,Sica A,Garlanda C,et al. The Yin Yan of tumorassociated macrophages in neoplastic progression and immune surveillance[J]. Immunol Rev,2008,222:155-161
[22] Burkholder B,Huang RY,Burgess R,et al. Tumor-induced perturbations of cytokines and immune cell networks[J]. Biochim Biophys Acta,2014,1845(2):182-201
[23] Colegio OR,Chu NQ,Szabo AL,et al. Functional polarization of tumour-associated macrophages by tumour-derived lactic acid[J].Nature,2014,513(7519):559-563
[24] Wang X,Luo G,Zhang K, et al. Hypoxic Tumor-Derived Exosomal miR-301a Mediates M2 Macrophage Polarization via PTEN/PI3Kγto Promote Pancreatic Cancer Metastasis[J].Cancer Res,78(16):4586-4598
[25] Cooks T,Pateras IS,Jenkins LM,et al. Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246[J]. Nat Commun,2018,9(1):771
[26] Ying X,Wu Q,Wu X,et al. Epithelial ovarian cancer-secreted exosomal miR-222-3p induces polarization of tumor-associated macrophages[J]. Oncotarget,2016,7(28):43076-43087