Mechanisms of Stromal Cell Activation by the Developing Tumor

现在人们认识到，肿瘤间质有助于肿瘤的发展，并可以促进肿瘤细胞从原发肿瘤向转移部位的迁移。组织学上，肿瘤有活跃的间质，这也是伤口愈合的标志。事实上，肿瘤被描述为无法愈合的伤口。创伤可激活参与修复过程的成纤维细胞，而创伤成纤维细胞也具有肿瘤周围成纤维细胞的一些特征，包括分泌生长因子(细胞因子、化学诱导剂)和基质蛋白的谱。有趣的是，伤口微环境对组织再生和基质干细胞和祖细胞的激活是高度允许的，因此也可能促进肿瘤干细胞库的扩张。在这个项目中，我们使用组织培养和动物模型来研究肿瘤和基质细胞之间的串扰。我们建立了一种同种异体原位小鼠模型，以研究创伤对乳腺癌进展的影响。在2007财年，我们利用裸鼠研究了T细胞室，并利用SMAD3基因敲除模型探讨了基质转化生长因子-β信号对创伤促进肿瘤生长的影响。我们目前正在研究SMAD3介导的血管生成在该模型中对肿瘤生长的贡献。为了研究人类癌症中基质细胞重编程的机制，我们与弗吉尼亚大学妇科肿瘤科建立了一个合作关系。我们从他们那里获得了来自子宫内膜癌及其周围正常子宫内膜的新鲜组织。我们使用这些样本来培养正常组织和癌症组织中的成纤维细胞，目的是通过RNA表达谱、蛋白质组学、microRNA分析来表征正常和癌症基质细胞之间的差异，以及在小鼠异种移植模型中对癌细胞生长的影响。今年，我们收集了7对正常/肿瘤来源的成纤维细胞，通过使用上皮/间充质标记来确定细胞群体的纯度，并对样本进行了microRNA图谱分析。初步数据显示，确实有一些microRNAs在正常成纤维细胞和肿瘤成纤维细胞中存在差异表达。我们现在正准备用实时定量聚合酶链式反应技术进一步验证这些结果。我们与Tim Veenstras博士团队(NCI-FCRF)建立了合作关系，并进行了初步实验，以优化分泌蛋白质的质谱分析条件。目前，我们正在制备SILAC试剂标记的细胞，用于正常或癌组织成纤维细胞分泌和细胞内蛋白的蛋白质组学研究。与梅琳达·霍林希德博士合作，我们正在开发一种测试方法，该方法将使我们能够测试子宫内膜间质细胞在支持小鼠肿瘤生长方面的作用。这一实验将是研究microRNA或其他差异表达基因功能的有价值的工具，这些基因可能有助于基质成纤维细胞的肿瘤生长促进功能。

It is now appreciated that the tumor stroma contributes to tumor development, and can facilitate migration of tumor cells from the primary tumor to metastatic sites. Histologically, tumors have an activated stroma which is also a hallmark of wound healing. Indeed, tumors have been described as wounds that do not heal. Wounding activates fibroblasts involved in the repair process, and wound fibroblasts are known to share several features also found to be present in peritumor fibroblasts including profiles of secreted growth factors (cytokines, chemoattractants) and matrix proteins. Interestingly, the wound microenvironment is highly permissive for tissue regeneration and activation of stromal stem and progenitor cells and may therefore also foster expansion of tumor stem cell pools. In this project we use tissue culture and animal models to study the crosstalk between tumor and stromal cells. We established a syngeneic orthotopic mouse model to investigate the influence of wounding on breast cancer progression. During fiscal year 2007 we explored the influence of the T-cell compartment using athymic nude mice, and of stromal TGF-beta signaling on wound promoted tumor growth by using a Smad3 knockout model. We are currently investigating the contribution of Smad3 mediated angiogenesis to tumor growth in this model. To investigate the mechanism of stromal cell reprogramming in human cancers we established a collaboration with the Division of Gynecologic Oncology at the University of Virginia. From them we have received fresh tissue from endometrial cancer and surrounding normal endometrium. We use those samples to grow fibroblasts from both the normal and cancerous tissue with the goal of characterizing the differences between normal and cancer stromal cells using RNA expression profiling, proteomics, microRNA analysis, and the effect on cancer cell growth in mouse xenograft models. This year we accumulated seven pairs of normal/tumor-derived fibroblasts, determined the purity of the cell population by using epithelial/mesenchymal markers and performed microRNA profiling on the samples. The preliminary data show that indeed some microRNAs were differentially expressed in normal versus cancer fibroblasts. We are now preparing for further validation of these results by quantitative real-time PCR technique. We established a collaboration with Dr. Tim Veenstras group (NCI-FCRF) and performed preliminary experiments to optimize the conditions for mass spectrometry analysis of secreted proteins. At present we are preparing cells labeled with the SILAC reagent that will be used for the proteomics study of secreted and intracellular proteins from fibroblasts derived from normal or cancer tissue. In collaboration with Dr. Melinda Holinshead we are developing an assay which will allow us to test the role of endometrial stromal cells in supporting tumor growth in mice. This assay will be a valuable tool to study the function of microRNA or other differentially expressed genes that may contribute to tumor growth promoting functions of stromal fibroblasts.