Novel method of identifying circulating mammary tumor cells

描述(由申请人提供)：建议项目的总体目标是严格开发识别、回收和分析活的循环肿瘤细胞(CTCs)的新方法。CTCs被认为是导致绝大多数癌症患者死亡的转移灶的前驱细胞，CTCs的分子和细胞分析可以为研究转移机制提供关键的信息。虽然目前有多种方法用于识别CTC，但大多数方法都是有限的，因为它们依赖于检测细胞表面上皮抗原的能力。上皮向间充质转化过程中上皮特性的丧失，以及促进转移的细胞表面糖蛋白干扰抗体识别细胞表面抗原的能力，引起了人们对这些过程的保真度的极大关注。在这里，我们将发展 基于癌细胞相对于未转化细胞的代谢状态的改变，识别CTC的新的、更稳健的方法。恶性细胞摄取葡萄糖和从细胞内环境中排出质子的能力的提高将被用作我们区分癌症和非癌症细胞的基础。在目标1中，我们将评估荧光葡萄糖衍生物和细胞内pH指示剂区分恶性和未转化细胞的能力。这些研究将在培养中的贴壁细胞和稀释到小鼠血液中的恶性细胞中进行。Aim 1的研究将确定，可以根据恶性肿瘤细胞的代谢特性将其与未转化细胞区分开来，并将开始制定在实验条件下鉴定血液中CTCs的必要方案。在目标2中，我们将把目标1中开发的技术应用于乳腺癌的小鼠模型。在这些研究中，我们将确定来自原位移植肿瘤的CTC是否可以使用代谢传感器进行识别，以及新开发的方法是否优于传统方法。我们预计，拟议研究的成功完成将为研究人员提供必要的工具，以便更可靠地从小鼠模型中识别和恢复CTCs，并最终证明对更准确地确定癌症患者血液中的CTCs是有益的。

DESCRIPTION (provided by applicant): The overall objective of the proposed project is to rigorously develop novel methods for identifying, recovering and analyzing viable circulating tumor cells (CTCs). CTCs are the presumed precursors of the metastatic lesions responsible for the deaths of the vast majority of carcinoma patients, and the molecular and cellular analysis of CTCs could provide key insight into mechanisms underlying metastasis. While there are a variety of methods that are currently employed for identifying CTCs, most are limited because of their reliance on the ability to detect cell surface epithelial antigens. The loss of epithelial character with the epithelial-to-mesenchymal transition, as well as the ability of metastasis-promoting cell surface glycoproteins to interfere with antibody recognition of cell surface antigens, raises significant concerns as to the fidelity of these procedures. Here we will develop novel, more robust methods for identifying CTCs based on the altered metabolic state of cancer cells relative to non-transformed cells. The elevated ability of malignant cells to take up glucos and to extrude protons from their intracellular environment will be used as the basis for our assays to distinguish cancer from non-cancer cells. In Aim 1 we will assess the abilities of a fluorescent glucose derivative and an intracellular pH indicator to distinguish between malignant and non-transformed cells. These studies will be carried out with both adherent cells in culture and with malignant cells diluted into mouse blood. The studies of Aim 1 will establish that malignant tumor cells can be distinguished from non-transformed cells based on their metabolic properties, and will begin to develop the protocols necessary to identify CTCs in blood under experimental conditions. In Aim 2 we will apply the techniques developed in Aim 1 to a mouse model of breast cancer. In these studies we will determine whether CTCs derived from orthotopically implanted tumors can be identified using the metabolic sensors, and whether the newly developed methods are superior to conventional approaches. We anticipate that the successful completion of the proposed studies will provide investigators with the tools necessary to more reliably identify and recover CTCs from mouse models, and may ultimately prove beneficial in more precisely defining CTCs in the blood of carcinoma patients.