Defining the formation and function of carcinoma-associated mesenchymal stem cells in the ovarian cancer microenvironment

摘要：明确肿瘤相关间充质干细胞的形成和功能。 卵巢癌的微环境 卵巢癌是美国最致命的妇科恶性肿瘤，死亡率在5岁时超过50% 好几年了。卵巢癌的特点是早期腹膜内转移和发展成复合体。 支持肿瘤细胞生长、存活和扩散的微环境。理解并最终 针对这种促进癌症的微环境提供了强大的新治疗方法的潜力 接近了。我的最终目标是成为一名研究卵巢的世界级独立内科科学家 癌症微环境，以开发新的治疗方法并改善卵巢癌妇女的预后 癌症。这个建议描述了重要的和创新的研究，这些研究将为我的未来奠定基础 除了提供对我的成功至关重要的必要技能和指导外，我的职业生涯也是如此。 卵巢肿瘤微环境(TME)是一个由多种细胞和化学成分组成的系统。这个 细胞性TME包括肿瘤细胞和非恶性间质细胞。化学物质TME的特征是酸中毒。 和缺氧。肿瘤相关间充质干细胞(CA-MSCs)是体内多潜能的基质细胞。 TME细胞可以分化为多种促肿瘤基质细胞类型，包括成纤维细胞， 肌成纤维细胞和脂肪细胞。CA-MSCs在基因上是正常的，没有恶性潜能，但 在功能上不同于正常组织或骨髓来源的MSCs。与正常MSCs相比，CA-MSCs 表现出独特的分子表型，具有非常高的骨形态发生蛋白(BMPs)表达。 由于这种独特的表型，这些CA-MSCs强烈促进卵巢癌的生长，增强 化疗耐药，并丰富肿瘤干细胞样群。 CA-MSCs是如何形成其独特表型的尚不清楚。我的初步数据显示肿瘤 分泌因子诱导了一些与CA-MSCs相关的分子变化。另一个潜在的调解人 CA-MSC的表型为低氧。低氧是已知的影响正常的化学物质TME的一个特征 MSC功能。在癌症中，低氧影响肿瘤：基质相互作用和低氧是BMP的关键调节因素 高水平表达是卵巢癌CA-MSCs的特征。初步数据显示， 低氧可增强肿瘤细胞在正常MSCs中诱导CA-MSC表达的能力。而当 这种诱导的机制尚不清楚，因为CA-MSCs在基因上是正常的，但保持其独特的 跨多代的表型，肿瘤诱导的表观遗传调节可能是形成 CA-MSC表型。事实上，初步数据表明，CA-MSCs表现出显著的低甲基化 与正常骨髓间充质干细胞相比。 低氧除了影响CA-MSC的形成外，还可能对CA-MSC的功能起关键的调节作用。 间充质干细胞已在卵巢TME中建立。我的初步数据显示，缺氧维持了 CA-MSCs的“干性”减缓生长并维持分化能力。此外，我的数据表明， 低氧诱导因子途径是CA-MSCs低氧信号传导的主要途径 与正常骨髓间充质干细胞相比。因此，低氧可能是卵巢TME内CA-MSCs的重要调节因子。 我的主要研究目标是了解CA-MSCs是如何获得其独特的表型并随后 与卵巢癌微环境相互作用并影响其功能。为了实现这一目标，我建议 两个具体目标： 1)测定正常MSCs获得CA-MSC样表型的能力 2)确定低氧对肿瘤微环境中已建立的CA-MSCs的影响。 在目标1中，我假设肿瘤细胞在低氧条件下诱导正常的MSCs成为CA-MSCs。 为了测试这一点，我将进行癌细胞：正常MSC在常氧和低氧下共培养，以确定 癌细胞可以从功能上将正常的MSC转变为CA-MSC。我还将探索差异DNA甲基化 作为创建CA-MSC的机制。在目标2中，我将重点介绍已经建立的CA-MSCs。我 假设低氧增强肿瘤内已建立的CA-MSCs的促肿瘤作用 微环境。为了测试这一点，我将使用条件性HIF途径基因敲除小鼠和CRISPER/Cas9 基因组编辑以评估缺氧和HIF信号对已建立的CA-MSCs的影响。 除了加深我们对CA-MSCs在卵巢癌中的理解外，拟议的研究和培训 将培养研究卵巢TME的独立职业所需的专业知识。通过…的支持 莱尔德博士，我将掌握全基因组表观遗传修饰的评估和大规模的分析 《组学》数据。Schipani博士将促进我在缺氧和HIF信号方面的教育。通过Schipani博士和 赵博士，我会学习制造和操作转基因小鼠模型。巴克诺维奇医生正在进行 指导将加深我在卵巢TME功能方面的专业知识，并与我的指导一起 这将有助于培养我的领导力、团队建设和沟通能力。到培训结束时 在此期间，我将开发一套新的技能集，它融合了多个科学领导者的专业知识 一位训练有素的内科科学家，是研究卵巢癌微环境的理想人选。

ABSTRACT: Defining the formation and function of carcinoma-associated mesenchymal stem cells in the ovarian cancer microenvironment Ovarian cancer is the most deadly US gynecologic malignancy with a mortality rate that exceeds 50% at 5 years. Ovarian cancer is characterized by early intraperitoneal metastasis and the development of a complex microenvironment which supports tumor cell growth, survival and spread. Understanding and eventually targeting this cancer-promoting tumor microenvironment offers the potential for powerful new therapeutic approaches. My ultimate goal is to become a world-class independent physician scientist studying the ovarian cancer microenvironment in order to develop new treatments and improve outcomes for women with ovarian cancer. This proposal describes important and innovative research which will lay the foundation for my future career in addition to providing the necessary skills and mentorship vital for my success. The ovarian tumor microenvironment (TME) is a diverse system of cellular and chemical components. The cellular TME includes tumor cells and non-malignant stromal cells. The chemical TME is marked by acidosis and hypoxia. Carcinoma-associated mesenchymal stem cells (CA-MSCs) are multi-potent stromal cells within the cellular TME that can differentiate into multiple pro-tumorigenic stromal cell types including fibroblasts, myofibroblasts, and adipocytes. CA-MSCs are genotypically normal without malignant potential but are functionally different than normal tissue or bone marrow derived MSCs. Compared to normal MSCs, CA-MSCs demonstrate a unique molecular phenotype with very high expression of bone morphogenetic proteins (BMPs). Due to this unique phenotype, these CA-MSCs strongly promote ovarian cancer growth, enhance chemotherapy resistance and enrich the cancer stem cell-like population. How CA-MSCs develop their unique phenotype remains unclear. My preliminary data indicate that tumor secreted factors induce some of the molecular changes associated with CA-MSCs. Another potential mediator of the CA-MSC phenotype is hypoxia. Hypoxia is a hallmark of the chemical TME known to impact normal MSC function. In cancer, hypoxia influences tumor:stromal interactions and hypoxia is a key regulator of BMP expression—high levels of which characterize ovarian cancer CA-MSCs. Preliminary data indicates that hypoxia enhances the ability of tumor cells to induce a CA-MSC expression profile in normal MSCs. While the mechanism of this induction is unknown, given CA-MSCs are genetically normal yet maintain their unique phenotype across multiple passages, tumor-induced epigenetic regulation may be critical to the formation of the CA-MSC phenotype. Indeed, preliminary data indicates CA-MSCs exhibit significant hypomethylation compared to normal MSCs. In addition to influencing the formation of a CA-MSC, hypoxia may also critically regulate the function of CA- MSCs already established in the ovarian TME. My preliminary data suggests that hypoxia maintains the “stemness” of CA-MSCs slowing growth and maintaining differentiation capacity. Further, my data suggests that the hypoxia inducible factor pathway, the main hypoxia signaling pathway, is hyper-active in CA-MSCs compared to normal MSCs. Thus hypoxia may be a critical modulator of CA-MSCs within the ovarian TME. My main research goal is to understand how CA-MSCs obtain their unique phenotype and subsequently interact with and influence the function of the ovarian cancer microenvironment. To achieve this goal, I propose two specific aims: 1) Determine the ability of normal MSCs to acquire a CA-MSC-like phenotype 2) Determine the impact of hypoxia on established CA-MSCs within the tumor microenvironment. In aim 1, I hypothesize that tumor cell conditioning under hypoxia induces normal MSCs to become CA-MSCs. To test this I will perform cancer cell: normal MSC co-cultures under normoxia and hypoxia to determine if cancer cells can functionally turn a normal MSC into a CA-MSC. I will also explore differential DNA methylation as a mechanism for the creation of a CA-MSC. In aim 2, I focus on already established CA-MSCs. I hypothesize that hypoxia enhances the pro-tumorigenic effects of established CA-MSCs within the tumor microenvironment. To test this, I will utilize conditional HIF pathway knockout mice and CRISPER/CAS9 genome editing to assess the impact of hypoxia and HIF signaling on established CA-MSCs. In addition to furthering our understanding of CA-MSCs in ovarian cancer, the proposed research and training will cultivate expertise necessary for an independent career studying the ovarian TME. Through the support of Dr. Laird, I will master the assessment of genome-wide epigenetic modifications and the analysis of large scale “omics” data. Dr. Schipani will facilitate my education in hypoxia and HIF signaling. Through Dr. Schipani and Dr. Cho, I will learn to generate and manipulate transgenic mouse models. Dr. Buckanovich's ongoing mentorship will further my expertise in the function of the ovarian TME and, together with my mentoring committee, will help develop my leadership, team-building and communication skills. By the end of the training period, I will have developed a novel skill set which merges the expertise of multiple scientific leaders yielding a uniquely trained physician scientist ideal for the study of the ovarian cancer microenvironment.