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

描述（由申请人提供）：研究原代人乳腺癌细胞内渗的新方法（PQ #24）。大约30-40%被诊断患有可治愈乳腺癌的女性最终死于转移性疾病。由于目前可用的技术来研究转移过程是有限的，迫切需要开发新的方法。我们一直在开发一种研究转移的内渗步骤的方法，该方法由3个综合方面组成：1）设计内渗测定（INA），2）优化从患者样本中分离原代人乳腺癌细胞的方法，3）采用多光子成像（MI）探索导致内渗的细胞相互作用。使用这些技术，我们将探索一个假设，即人类乳腺癌含有不同比例的具有不同基因表达谱的具有浸润能力的癌细胞，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）我们可以用INA来研究乳腺癌细针穿刺活检（FNA）获得的细胞的内渗活性：2）人乳腺癌中有不同比例的内渗活性细胞，并且在巨噬细胞存在下内渗更好; 3）具有内渗能力的原代人乳腺癌细胞表达与内渗特征相关的肌动蛋白调节蛋白Mena的独特同种型; 4）具有高MenaINV和低Menalla同种型表达模式的癌细胞与巨噬细胞一起参与体内乳腺肿瘤中的跨内皮迁移和内渗，如通过INA评估的。5）在人乳腺癌中存在被称为TMEM的内渗位点，其含有过表达Mena的肿瘤细胞和巨噬细胞，并且TMEM位点的数量与远处转移相关。我们也有证据表明，一些内渗能力的细胞粘附在顶端内皮表面后，内渗，而其他人没有。我们建议使用INA和MI来研究人乳腺癌细胞与内皮细胞和巨噬细胞在INA中浸润时的空间和时间相互作用，并评估雌激素、Her 2Neu受体和Mena同种型在浸润感受态细胞中的表达模式。此外，我们将把临床病理学参数与在血管内浸润时粘附于内皮顶端表面的血管内浸润活性细胞的百分比以及不粘附于内皮顶端表面的血管内浸润活性细胞的百分比相关联，评估Mena亚型过表达对血管内浸润的影响，并定义一组在血管内浸润活性人乳腺癌细胞中独特表达的基因。为了评估癌细胞在体外交叉工程化内皮的能力是否反映了其体内的内渗潜力，我们将收集荧光标记的内渗活性细胞和无内渗活性细胞，以确定与内皮的差异相互作用，鉴定参与内皮相互作用的基因，并在体外和体内实验性地操纵其内渗活性。 公共卫生相关性：研究原发性人乳腺癌细胞内渗的新方法（PQ#24）。这种新方法将使我们能够研究与人类疾病最相关的内渗生物学，因为我们将使用从患者乳腺癌切除术中获得的人类乳腺癌细胞。此外，它将使我们能够定义人类内渗特征，这将代表开发抗转移治疗靶点的基础，这些靶点迫切需要降低乳腺癌相关的发病率和死亡率。

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. PUBLIC HEALTH RELEVANCE: A new approach for studying intravasation of primary human breast cancer cells (PQ#24). The new approach will allow us to study the biology of intravasation most relevant to human disease because we will use human breast cancer cells obtained from patients' breast cancer excisions. In addition, it will allow us to define human intravasation signature which will represent a foundation for development of anti-metastatic therapeutic targets which are urgently needed to decrease breast cancer associated morbidity and mortality.