SILAC–based quantitative MS approach for real-time recording protein-mediated cell-cell interactions

被引:0
作者
Xixi Wang
Yu He
Yang Ye
Xinyu Zhao
Shi Deng
Gu He
Hongxia Zhu
Ningzhi Xu
Shufang Liang
机构
[1] West China Hospital,State Key Laboratory of Biotherapy and Cancer Center
[2] Sichuan University,Department of Urinary Surgery
[3] and National Collaborative Innovation Center for Biotherapy,Laboratory of Cell and Molecular Biology & State Key Laboratory of Molecular Oncology
[4] Chengdu Center for Disease Control and Prevention,undefined
[5] West China Hospital,undefined
[6] West China Medical School,undefined
[7] Sichuan University,undefined
[8] Cancer Institute & Cancer Hospital,undefined
[9] Chinese Academy of Medical Sciences,undefined
来源
Scientific Reports | / 8卷
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摘要
In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. To understand the molecular mechanisms of these complex interplays, the secreted protein analysis between malignant cancer cells and the surrounding nonmalignant stroma is a good viewpoint to investigate cell-cell interactions. Here, we developed two stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS)/MS approaches termed spike-in SILAC and triple-SILAC to quantify changes of protein secretion level in a cell co-cultured system. Within the co-culture system of CT26 and Ana-1 cells, the spike-in SILAC and triple-SILAC MS approaches are sensitive to quantitatively measure protein secretion changes. Three representative quantified proteins (Galectin-1, Cathepsin L1 and Thrombospondin-1) by two SILAC-based MS methods were further validated by Western blotting, and the coming result matched well with SILACs’. We further applied these two SILACs to human cell lines, NCM460 and HT29 co-culture system, for evaluating the feasibility, which confirmed the spike-in and triple SILAC were capable of monitoring the changed secreted proteins of human cell lines. Considering these two strategies in time consuming, sample complexity and proteome coverage, the triple-SILAC way shows more efficiency and economy for real-time recording secreted protein levels in tumor microenvironment.
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共 40 条
[1]  
Clementz AG(2013)Collagen XV: exploring its structure and role within the tumor microenvironment Mol Cancer Res. 11 1481-1486
[2]  
Sangaletti S(2017)The good and bad of targeting cancer-associated extracellular matrix Curr Opin Pharmacol. 35 75-82
[3]  
Slany A(2015)Targeting breast cancer-associated fibroblasts to improve anti-cancer therapy Breast. 24 532-538
[4]  
Giussani M(2015)Tumor-extracellular matrix interactions: Identification of tools associated with breast cancer progression Semin Cancer Biol. 35 3-10
[5]  
Kostourou V(2014)Non-collagenous ECM proteins in blood vessel morphogenesis and cancer Biochim Biophys Acta. 1840 2403-2413
[6]  
Zeng X(2013)Quantitative secretome analysis reveals the interactions between epithelia and tumor cells by J Proteomics. 89 51-70
[7]  
Zhong L(2008) modulating colon cancer microenvironment Cancer Res. 68 7237-7245
[8]  
Li M(2010)Identification of secreted proteins that mediate cell-cell interactions in an J Biol Chem. 285 6285-6297
[9]  
Li X(2016) model of the lung cancer microenvironment Exp Hematol. 44 1059-1071
[10]  
Liang S(2009)Intercellular transfer of proteins as identified by stable isotope labeling of amino acids in cell culture Curr Proteomics. 6 25-31