Developing Adhesome Technology as a Physical Marker of Highly Metastatic Cells

项目摘要 诊断标记物提高了我们在早期阶段识别肿瘤的能力，但继发性转移 仍然非常普遍且具有攻击性转移是由于原发性肿瘤脱落小的 干细胞样细胞的亚群，可以迁移通过相邻的基质和整个扩散 与原发肿瘤中残留的细胞不同。分子预后因子的研究进展 可以评估转移风险的这些干细胞样细胞的标记物是有限的;然而最近， 有证据表明，肿瘤的物理状态可以作为另一种预后标志物。我们有 观察到一个这样的物理标记-粘附强度-在转移性上皮细胞系中从 乳腺、前列腺和肺肿瘤由于在基质样条件下粘着斑组装的差异， 例如低Mg+2和Ca+2。因此，强烈粘附的亚种群比它们的亚种群更慢， 同行。我们的假设是，匡威的群体，即弱粘附的肿瘤细胞在肿瘤细胞中， 间质，代表最具转移性的亚群，其可基于其粘附强度进行纯化 并计数以确定转移风险。为了分离这种匡威的弱粘附细胞群，我们 已经开发了一种“粘附体”流动室测定法，其将使细胞经受低剪切（<10达因/cm 2）， 基质样环境，并快速（<10分钟）分选107个细胞。使用该技术进行概念验证， 目的1，我们将验证其分选效率和保真度，并观察弱贴壁细胞的潜力， 分离、迁移和侵入类似于邻近肿瘤间质的工程微环境。在目标2中， 将测试粘附体流动室预测乳腺癌转移风险的能力， 选择的细胞进入乳房脂肪垫。然后，我们将评估弱或强粘附细胞的倾向 形成或不形成转移。将直接评估来自原发性肿瘤间质的细胞， 鉴定具有弱粘附特征的细胞，并将其与继发性转移形成相关联， 发生在诊所。通过验证肿瘤类型，这种创新的分子和细胞分析 技术R21提案将建立粘附体流动室技术并验证我们的假设 弱粘附的肿瘤细胞能够迁移并形成继发性转移，即建立 转移风险因此，这项技术应该为临床医生提供额外的肿瘤预后评估。 转移风险

Project Summary Diagnostic markers have improved our ability to identify tumors at earlier stages, but secondary metastases are still exceedingly common and aggressive. Metastases result from primary tumors shedding a small subpopulation of stem-like cells that can migrate through adjacent stroma and disseminate throughout the body unlike those cells remaining in the primary tumor. Progress towards identifying molecular prognostic markers of these stem-like cells that could assess metastatic risk has been limited; very recently however, evidence has suggested that a tumor's physical state may act as an alternative prognostic marker . We have observed that one such physical marker–adhesion strength–varies in metastatic epithelial cell lines from mammary, prostate, and lung tumors due to differences in focal adhesion assembly in stromal-like conditions, e.g. low Mg+2 and Ca+2. Thus strongly adherent sub-populations are slower and less processive than their unselected counterparts. Our hypothesis is that the converse population, i.e. weakly adherent tumor cells in the stroma, represents the most metastatic sub-population, which can be purified based on their adhesion strength and counted to determine metastatic risk. To isolate this converse population of weakly adherent cells, we have developed an “adhesome” flow chamber assay that will subject cells to low shear (<10 dynes/cm2) in stromal-like environments and rapidly (<10 min) sort 107 cells. Using this technology for proof-of-concept in Aim 1, we will validate its sorting efficiency and fidelity and observe the potential of weakly adherent cells to detach, migrate, and invade engineered microenvironments that resemble adjacent tumor stroma. In Aim 2, we will test the adhesome flow chamber's ability to predict breast cancer metastatic risk in vivo by injecting selected cells into mammary fat pads. We will then assess the propensity of weakly or strongly adherent cells to form or not form metastases, respectively. Cells from primary tumor stroma will be directly assessed to identify cells with a weakly adhesive signature and correlate that with secondary metastasis formation as would occur in the clinic. By validating across tumor types, this Innovative Molecular and Cellular Analysis Technologies R21 proposal will establish an adhesome flow chamber technology and validate our hypothesis that weakly adherent tumor cells are capable of migrating and forming secondary metastases, i.e. establish metastatic risk. Thus this technology should provide clinicians with addition prognostic assessment of a tumor's metastatic risk.