BIO-POD: PALLET MICRO-ARRAY FOR RARE CELL ANALYSIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们已经开发了一种托盘阵列纳米技术，允许分离单个贴壁细胞。 将这种纳米技术与基于抗体的检测方法、先进的光学成像和乳腺肿瘤的细针抽吸（FNA）取样相结合，我们正在试验一种方法，该方法1）克服了现有技术的局限性，如激光显微切割，2）可以在诊断时与肿瘤切除后提供给患者，3）允许对肿瘤内存在的各种细胞成分进行计数，可能产生用于预后或预测治疗功效的重要信息，4）设计用于高通量自动化分析，和4）具有评估个体细胞区室的分子谱的潜力，这可以为定制的个体化治疗的设计提供额外的信息。 这些研究的假设是托盘阵列纳米技术将允许从原发性乳腺肿瘤中识别、计数和分离以下单个细胞成分：乳腺癌干细胞、内皮祖细胞、肌上皮细胞和炎性浸润。 将通过追求以下具体目标来检验这一假设： 1.建立鉴别罕见贴壁细胞的检测阈值。 我们将采用表达不同比例的细胞表面分子的独特组合的细胞混合物，通过流式细胞术确认，应用于托盘阵列。 由于多个肿瘤细胞亚群的检测将需要荧光/多抗原检测，我们将进行多色荧光成像，以建立罕见细胞的检测阈值。 此外，我们将完善托盘阵列的设计。 2.使用细针抽吸（FNA）取样将此方法应用于原发性乳腺癌细胞。 将使用切除后立即进行的FNA对乳腺肿瘤标本进行初步可行性研究，以测试将结果从细胞系推广到原发性肿瘤组织的能力。 这些研究将建立乳腺癌干细胞、内皮祖细胞、肌上皮细胞、肿瘤上皮细胞和炎性浸润的分离、鉴定和检测方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a pallet array nanotechnology that permits isolation of individual adherent cells. Combining this nanotechnology with antibody-based detection methods, advanced optical imaging and fine needle aspirate (FNA) sampling of breast tumors, we are piloting a methodology that 1) overcomes limitations to existing technologies, such as laser microdissection, 2) could be available to patients at the time of diagnosis vs. after tumor resection, 3) permits enumeration of various cellular elements present within a tumor potentially yielding important information for prognosis or predictive of therapeutic efficacy, 4) is designed for high throughput automated analyses, and 4) has the potential to assess the molecular profile of individual cellular compartments, which could provide additional information for the design of tailored individualized therapy. The HYPOTHESIS for these studies is that the pallet array nanotechnology will permit the identification, enumeration, and isolation of the following individual cellular elements from primary breast tumors: breast cancer stem cells, endothelial progenitor cells, myoepithelial cells, and inflammatory infiltrate. This hypothesis will be tested by pursuing the following Specific Aims: 1. Establish the detection threshold for identifying rare adherent cells. We will employ mixtures of cells expressing unique combinations of cell surface molecules in varying proportions, confirmed by flow cytometry, applied to the pallet array. As detection of multiple tumor cellular subsets will require multicolor/multi-antigen detection, we will perform multi-color fluorescence imaging to establish the detection threshold for rare cells. Additionally, we will refine the design of the pallet array. 2. Apply this methodology to primary breast cancer cells using Fine Needle Aspirate (FNA) sampling. Pilot feasibility studies will be conducted on breast tumor specimens using FNAs performed immediately after resection, in order to test the ability to generalize the findings from cell lines to primary tumor tissues. These studies will establish isolation, identification, and detection methods for the enumeration of breast cancer stem cells, endothelial progenitor cells, myoepithelial cells, tumor epithelial cells, and inflammatory infiltrates.