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

摘要：确定肿瘤相关间充质干细胞的形成和功能， 卵巢癌微环境 卵巢癌是美国最致命的妇科恶性肿瘤，死亡率超过50%， 年卵巢癌的特点是早期腹膜内转移和复杂的发展， 微环境支持肿瘤细胞生长、存活和扩散。理解并最终 以这种促癌肿瘤微环境为靶点， 接近。我的最终目标是成为一名世界级的独立医生科学家， 癌症微环境，以开发新的治疗方法，改善卵巢癌妇女的预后。 癌这份提案描述了重要的创新研究，这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之外，我还提供了职业生涯。 卵巢肿瘤微环境（TME）是一个由细胞和化学成分组成的多样化系统。的 细胞TME包括肿瘤细胞和非恶性基质细胞。化学TME的特征是酸中毒 和缺氧。癌相关间充质干细胞（CA-MSCs）是肿瘤内的多能基质细胞， 细胞TME可以分化成多种促肿瘤发生基质细胞类型，包括成纤维细胞， 肌成纤维细胞和脂肪细胞。CA-MSC基因型正常，无恶性潜能，但 在功能上不同于正常组织或骨髓来源的MSC。与正常MSCs相比，CA-MSCs 显示具有非常高的骨形态发生蛋白（BMP）表达的独特分子表型。 由于这种独特的表型，这些CA-MSC强烈促进卵巢癌生长，增强卵巢癌细胞的增殖。 化疗耐药性和丰富癌症干细胞样群体。 CA-MSC如何发展其独特的表型仍不清楚。我的初步数据显示 分泌因子诱导一些与CA-MSC相关的分子变化。另一个潜在的调解人 CA-MSC表型的主要特征是缺氧。缺氧是化学TME的标志，已知会影响正常的 MSC功能。在癌症中，缺氧影响肿瘤：基质相互作用和缺氧是BMP的关键调节因子 高水平表达是卵巢癌CA-MSC的特征。初步数据显示， 缺氧增强肿瘤细胞在正常MSC中诱导CA-MSC表达谱的能力。而 这种诱导的机制是未知的，因为CA-MSC在遗传上是正常的，但仍保持其独特的 表型跨越多个传代，肿瘤诱导的表观遗传调控可能是至关重要的形成 CA-MSC表型。事实上，初步数据表明CA-MSC表现出显著的低甲基化， 与正常的MSC相比。 除了影响CA-MSC的形成外，缺氧还可能严重调节CA-MSC的功能。 间充质干细胞已经在卵巢TME中建立。我的初步数据显示缺氧可以维持 CA-MSC的“干性”减缓生长并维持分化能力。而且我的数据显示 缺氧诱导因子途径，主要的缺氧信号传导途径，在CA-MSCs中过度活跃， 与正常的MSC相比。因此，缺氧可能是卵巢TME内CA-MSC的关键调节剂。 我的主要研究目标是了解CA-MSCs如何获得其独特的表型， 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)确定正常MSC获得CA-MSC样表型的能力 2)确定缺氧对肿瘤微环境中已建立的CA-MSC的影响。 在目的1中，我假设肿瘤细胞在缺氧条件下诱导正常MSC成为CA-MSC。 为了测试这一点，我将在常氧和缺氧下进行癌细胞：正常MSC共培养，以确定是否 癌细胞可以在功能上将正常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，我专注于已经建立的CA-MSC。我 假设缺氧增强了肿瘤内已建立的CA-MSC的促肿瘤发生作用 微环境为了验证这一点，我将利用条件性HIF通路敲除小鼠和CRISPER/CAS9 基因组编辑以评估缺氧和HIF信号传导对已建立的CA-MSC的影响。 除了进一步了解CA-MSCs在卵巢癌中的作用外， 将培养必要的专业知识，为研究卵巢TME的独立职业生涯。通过支持 博士莱尔德，我将掌握全基因组表观遗传修饰的评估和大规模的分析， “omics”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 博士周，我将学习如何制作和操作转基因小鼠模型。Buckanovich医生正在 导师将进一步我的专业知识在卵巢TME的功能，并与我的导师一起， 委员会，将有助于发展我的领导能力，团队建设和沟通技巧。在培训结束时 在此期间，我将开发出一套新颖的技能，融合了多位科学领导者的专业知识， 一位受过独特训练的医生科学家，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.