Mechanisms of Stromal Cell Activation by the Developing Tumor

现在认识到，肿瘤间质有助于肿瘤发展，并且可以 促进肿瘤细胞从原发肿瘤迁移到转移部位。 在组织学上，肿瘤具有活化的基质，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。伤人 激活参与修复过程的成纤维细胞，并且已知伤口成纤维细胞共享 还发现在瘤周成纤维细胞中存在几个特征，包括 分泌的生长因子（细胞因子、化学引诱物）和基质蛋白。有趣的是 伤口微环境高度允许组织再生和基质的活化 因此也可以促进肿瘤干细胞库的扩增。在 本项目我们使用组织培养和动物模型来研究肿瘤和 基质细胞我们建立了一个同基因原位小鼠模型来研究 创伤对乳腺癌进展的影响在2007年财政年度，我们探讨了 使用无胸腺裸鼠的T细胞区室，以及伤口上的基质TGF-β信号传导 通过使用Smad 3敲除模型促进肿瘤生长。我们目前正在调查 在该模型中，Smad 3介导的血管生成对肿瘤生长的贡献。探讨 基质细胞重编程在人类癌症中的机制，我们与 弗吉尼亚大学妇科肿瘤学分部。我们从他们那里得到了 子宫内膜癌和周围正常子宫内膜的新鲜组织。我们用这些样本 从正常组织和癌组织中培养成纤维细胞， 使用RNA表达谱分析正常和癌症基质细胞之间的差异， 蛋白质组学、microRNA分析以及对小鼠异种移植模型中癌细胞生长的影响。 今年，我们积累了七对正常/肿瘤来源的成纤维细胞，确定了它们的细胞分化。 通过使用上皮/间充质标志物和进行的microRNA来纯化细胞群 对样本进行分析初步数据显示，确实有一些microRNA 在正常与癌成纤维细胞中差异表达。我们现在正准备进一步 通过定量实时PCR技术验证这些结果。我们建立了一个 与Tim Veenstras博士小组（NCI-FCRF）合作，并进行了初步研究 实验以优化分泌蛋白质的质谱分析条件。在 目前，我们正在制备用SILAC试剂标记的细胞， 正常人成纤维细胞分泌和细胞内蛋白质组学研究 或癌组织。我们与梅林达·霍林斯黑德博士合作， 这将使我们能够测试子宫内膜间质细胞在支持肿瘤生长中的作用， 对小鼠该检测方法将成为研究microRNA或其他生物学功能的有价值的工具。 差异表达的基因可能有助于肿瘤生长促进功能， 基质成纤维细胞

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.