State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, PR China.
J Proteomics. 2013 Aug 26;89:51-70. doi: 10.1016/j.jprot.2013.05.032. Epub 2013 Jun 5.
In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. Secreted proteins are responsible for crosstalk among these cells within tumor microenvironment. To elucidate the interactions of tumor and epithelia, we co-cultured colon cancer cell line HT29 with normal human colon mucosal epithelial cell line NCM460 to mimic tumor microenvironment in vitro and investigated the differential expression pattern of secretome. A quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture (SILAC) and LC-mass spectrometry was used for secretome analysis. Totally 45 proteins were altered over 2-fold in co-cultured cellular supernatants between equal amounts of NCM460 and HT29 cells, compared with mono-cultured conditions. These differential secreted proteins involve in multiple tumor-associated biological functions. The secretion level and acting pattern of acrogranin, IGFBP6 and vimentin were changed along with different co-cultured cell number ratios between NCM460 and HT29 cells, simulating early, middle or advanced stage of colon cancer. Therefore, a quantitative secretome profiling based on a co-culture system can track secreted protein changes and their associated biological roles between tumor and epithelia, which gives a new insight on communications between tumor and epithelia as well as cancer biotherapy by inhibiting cell interactions.
Tumor microenvironment is a complex system and comprised of cancer cells and host stromal cells. The growth and progression of tumor have been recognized were affected by multidirectional interactions of secreted proteins (secretome), which were produced by the cells within tumor microenvironment. Focus on general secreted molecules of living cells via proteomic tools, is promising for investigating cell communication. Stable isotope labeling by amino acids in cell culture (SILAC) is a metabolic labeling strategy for quantitative analysis, which is gaining popularity because of its ease of implementation, the high quality of quantitative data obtained, robustness and compatibility with existing experimental workflows. Therefore, SILAC-based quantitative secretome analysis was employed for investigating interactions between epithelia and tumor by in vitro modulating colon cancer microenvironment with established co-culture system, which simplified the complexity of cancer microenvironment, also tracked secreted protein changes and their associated biological roles between epithelia and cancer cells. A series of tumor associated secreted proteins was quantitated and investigated in our study. So, the results give a new insight on communications between tumor and epithelia as well as cancer biotherapy by inhibiting interactions of them.
在肿瘤微环境中，多种细胞类型之间的相互作用对癌症的进展至关重要。分泌蛋白负责肿瘤微环境中这些细胞之间的串扰。为了阐明肿瘤和上皮细胞的相互作用，我们将结肠癌细胞系 HT29 与正常人结肠黏膜上皮细胞系 NCM460 共培养，以在体外模拟肿瘤微环境，并研究分泌组的差异表达模式。基于稳定同位素标记氨基酸在细胞培养中的定量蛋白质组学方法（SILAC）和 LC-质谱法用于分泌组分析。与单培养条件相比，在等数量的 NCM460 和 HT29 细胞之间的共培养细胞上清液中，有 45 种蛋白质的表达水平发生了 2 倍以上的变化。这些差异分泌的蛋白质涉及多种与肿瘤相关的生物学功能。随着 NCM460 和 HT29 细胞之间共培养细胞数量比例的不同，颗粒蛋白、IGFBP6 和波形蛋白的分泌水平和作用模式发生变化，模拟结肠癌的早期、中期和晚期。因此，基于共培养系统的定量分泌组谱分析可以跟踪肿瘤和上皮细胞之间分泌蛋白的变化及其相关的生物学作用，为肿瘤和上皮细胞之间的通讯以及通过抑制细胞相互作用进行癌症生物治疗提供新的见解。
肿瘤微环境是一个复杂的系统，由肿瘤细胞和宿主基质细胞组成。肿瘤的生长和进展已被认为受到肿瘤微环境中细胞分泌的蛋白质（分泌组）的多向相互作用的影响。通过蛋白质组学工具关注活细胞的一般分泌分子，有望用于研究细胞通讯。稳定同位素标记氨基酸在细胞培养中的（SILAC）是一种定量分析的代谢标记策略，由于其易于实施、获得的定量数据质量高、稳健性以及与现有实验工作流程的兼容性，因此越来越受欢迎。因此，我们通过建立共培养系统体外调节结肠癌细胞微环境，采用 SILAC 基于定量的分泌组分析来研究上皮细胞与肿瘤之间的相互作用，简化了癌症微环境的复杂性，还跟踪了上皮细胞与癌细胞之间分泌蛋白的变化及其相关的生物学作用。在我们的研究中，对一系列与肿瘤相关的分泌蛋白进行了定量和研究。因此，这些结果为肿瘤和上皮细胞之间的通讯以及通过抑制它们的相互作用进行癌症生物治疗提供了新的见解。
