Novel approach to study intravasation in primary human breast cancer cells(PQ24)

描述(由申请人提供)：一种研究原代人类乳腺癌细胞血管内渗的新方法(PQ#24)。被诊断为可治愈的乳腺癌的女性中，大约有30%-40%最终死于转移性疾病。由于目前可用于研究转移过程的技术有限，迫切需要开发新的方法。我们一直在发展一种研究转移的血管内步骤的方法，包括三个综合方面：1)设计血管内试验(INA)，2)优化从患者样本中分离原代人乳腺癌细胞的方法，3)应用多光子成像(MI)来探索导致血管内转移的细胞相互作用。使用这些技术，我们将探索一种假设，即人类乳腺癌包含不同比例的具有不同基因表达谱的血管内能力癌细胞，它们决定了特定乳腺癌的临床行为。我们的初步研究表明：1)我们可以使用INA来研究从患者肿瘤细针抽吸活检(FNA)获得的乳腺癌细胞的血管内活性；2)人类乳腺癌具有不同比例的血管内活性细胞，并且在巨噬细胞存在的情况下血管内活性更好；3)血管内活性原代人类乳腺癌细胞表达与血管内信号相关的肌动蛋白调节蛋白MENA的独特亚型；4)高MenaINV和低Mena11a亚型表达的癌细胞与巨噬细胞共同参与乳腺肿瘤内皮细胞的跨内皮细胞迁移和体内血管内皮细胞的侵袭；5)乳腺癌中存在肿瘤细胞和巨噬细胞高表达的Mena的血管内皮细胞部位，TMEM部位的数量与远处转移有关。我们也有证据表明，一些血管内皮细胞在血管内皮细胞表面黏附，而另一些则不能。我们建议使用INA和MI来研究人乳腺癌细胞与血管内皮细胞和巨噬细胞在INA中的时空相互作用，并评估雌激素、HER2Neu受体和MENA异构体在血管内活性细胞中的表达模式。此外，我们将把临床病理参数与血管内皮细胞在血管内皮细胞根尖表面黏附的百分比相关联，评估Mena异构体过表达对血管内皮细胞的影响，并确定一组在血管内血管功能细胞中唯一表达的基因。为了评估癌细胞在体外跨越工程化内皮的能力是否反映了它们在体内的血管内皮细胞潜能，我们将收集荧光标记的血管内皮细胞和非血管内皮细胞，以确定与内皮细胞的不同相互作用，识别与内皮相互作用相关的基因，并在体外和体内实验操纵它们的血管内皮细胞活动。

DESCRIPTION (provided by applicant): A new approach for studying intravasation of primary human breast cancer cells (PQ#24). Approximately 30-40% of women diagnosed with a curable breast cancer eventually die of metastatic disease. Since currently available technologies to study metastatic process are limited there is an urgent need to develop new approaches. We have been developing an approach for studying the intravasation step of metastasis composed of 3 integrated aspects: 1) devising an intravasation assay (INA), 2) optimizing methods for primary human breast cancer cells isolation from patient samples, and 3) adapting multiphoton imaging (MI) to explore cellular interactions leading to intravasation. Using these technologies we will explore a hypothesis that human breast cancers contain varying proportions of intravasation-competent cancer cells with distinct gene expression profile and they determine the clinical behavior of a particular breast cancer. Our preliminary studies indicate that: 1) we can use INA to study intravasation activity of breast cancer cells obtained by fine needle aspiration biopsy (FNA) from patients' tumors; 2) human breast cancers have varying proportions of intravasation-competent cells, and intravasate better in the presence of macrophages; 3) intravasation- competent primary human breast cancer cells express unique isoforms of an actin regulatory protein Mena that are related to an intravasation signature; 4) cancer cells with high MenaINV and low Mena11a isoform expression pattern participate with macrophages in transendothelial migration as assessed by INA and intravasation in mammary tumors in vivo, 5) intravasation sites called TMEM containing Mena overexpressing tumor cells and macrophages exist in human breast carcinomas and the number of TMEM sites correlates with distant metastasis. We also have evidence that some intravasation-competent cells adhere to apical endothelial surface upon intravasation while others do not. We propose to use INA and MI to study spatial and temporal interactions of human breast cancer cells with endothelial cells and macrophages as they intravasate in INA and assess estrogen, Her2Neu receptor and Mena isoform expression pattern in the intravasation- competent cells. In addition, we will correlate th clinicopathological parameters with the percentage of intravasation-competent cells that adhere to endothelial apical surface upon intravasation and those that do not, assess the effect of Mena isoform overexpression on intravasation and define a set of genes uniquely expressed in intravasation-competent human breast cancer cells. To assess if the ability of cancer cells to cross engineered endothelium in vitro reflects their in vivo intravasation potential we will collec fluorescent- labeled intravasation-competent and incompetent cells to determine differential interactions with endothelia, identify genes involved in endothelial interactions, and experimentally manipulate their intravasation activities in vitro and in vivo.