Mechanisms of linkage of stem and invasive phenotypes during metastatic colonization

摘要 转移是乳腺癌相关死亡率的主要原因，是一个多步骤的过程， 在远处器官内形成肿瘤病灶。然而，在原发灶中只有癌细胞亚群 肿瘤微环境能够完成整个转移级联，包括内渗， 循环中的存活、外渗和远处肿瘤生长。我们发现了一群 侵袭性、非增殖性、非凋亡性、能够内渗的化学抗性癌细胞（已发表）， （初步结果）。这些细胞表达高水平的MenaINV，MenaINV是一种促转移的同种型。 肌动蛋白调节蛋白Mena需要成熟的侵入性突起称为invadopodia，这使得 癌细胞穿过内皮并扩散。MenaINV定位于细胞膜，这是必需的 其功能可能受PI 3 K β通过磷脂酰肌醇信号通路调节。我们发现MenaINV 通过直接接触在肿瘤细胞中诱导表达（已发表）和干细胞程序（初步结果） 肿瘤相关巨噬细胞。有趣的是，我们还发现化疗共诱导MenaINV和 干细胞程序以巨噬细胞依赖的方式。表达MenaINV的干细胞的出现可能 是转移的关键步骤之一，因为这些细胞不仅具有跨内皮迁移能力 还具有肿瘤引发能力。在原发性乳腺肿瘤中，表达高水平MenaINV的癌细胞 能够通过肿瘤转移微环境（TMEM）入口进入血管。这些紧密 我们的小组首先描述了毛细血管壁中受控的瞬时开口， 巨噬细胞、内皮细胞和表达Mena的肿瘤细胞直接物理接触。TMEM门道 在肺转移瘤中也观察到来自肺转移瘤的癌细胞再传播，但目前 尚不清楚是否需要干细胞程序才能从该次要部位传播癌细胞。有趣的是，我们和 其他人发现化疗以巨噬细胞依赖的方式增加了癌细胞的比例， 共表达高水平的MenaINV和干细胞转录因子S 0X 9。因此，我们假设， 在癌细胞中诱导跨内皮迁移能力表型（通过表达 MenaINV）在机制上与干细胞程序相关，并且这种跨内皮迁移的诱导- 有能力的茎表型通过化疗而增强。我们将使用高分辨率活体成像技术， 肺与干细胞和Mena生物传感器组合，以在体内确定干细胞和Mena的相互作用如何。 MenaINV调节肺的外渗和转移性定植，并确定其机制， 化疗诱导的缺氧和巨噬细胞流入影响肺转移灶中的癌症表型。 这一项目的成功完成将增进我们对以下方面所涉机制的了解： 远端转移的发展，并为发展所需的抗转移疗法提供基础， 提高转移性乳腺癌患者的生存率。

ABSTRACT Metastasis, the primary cause of breast cancer-related mortality, is a multistep process culminating with the formation of tumor foci within distant organs. However, only a subpopulation of cancer cells within the primary tumor microenvironment is capable of completing the entire metastatic cascade, which includes intravasation, survival in circulation, extravasation, and tumor growth at distant sites. We identified a population of highly invasive, non-proliferating, non-apoptotic, chemo-resistant cancer cells capable of intravasation (published) and extravasation (preliminary results). These cells express high levels of MenaINV, a pro-metastatic isoform of the actin-regulatory protein Mena required for maturation of invasive protrusions called invadopodia, which enable cancer cells to cross endothelium and disseminate. MenaINV localization to the cell membrane, which is required for its function, may be regulated by PI3Kβ through phosphoinositide signaling. We found that MenaINV expression (published) and a stem cell program (preliminary results) are induced in tumor cells by direct contact with tumor-associated macrophages. Interestingly, we also found that chemotherapy co-induces MenaINV and the stem program in a macrophage-dependent manner. The emergence of MenaINV expressing stem cells may be one of the crucial steps to metastasis because these cells are not only transendothelial migration-competent but also have tumor-initiating capability. In primary breast tumors, cancer cells expressing high levels of MenaINV are able to enter blood vessels through Tumor Microenvironments of Metastasis (TMEM) doorways. These tightly controlled transient openings in capillary walls were first described by our group and are composed of macrophages, endothelial cells and Mena-expressing tumor cells in direct physical contact. TMEM doorways and cancer cell re-dissemination from lung metastases are also observed in lung metastases, but it is currently unknown if stem program is required for cancer cell dissemination from this secondary site. Interestingly, we and others found that chemotherapy in a macrophage-dependent manner increases the proportion of cancer cells co-expressing high levels of MenaINV and stem cell transcription factor SOX9. Thus, we hypothesize that induction of the transendothelial migration-competent phenotype in cancer cells (identified by the expression of MenaINV) is mechanistically linked to the stem program, and that the induction of this transendothelial migration- competent stem phenotype is potentiated by chemotherapy. We will use high resolution intravital imaging of the lungs in combination with stem and Mena biosensors to determine in vivo how the interplay of stemness and MenaINV regulate extravasation and metastatic colonization of the lungs and determine the mechanism by which chemotherapy induced hypoxia and macrophage influx affect cancer phenotype in metastatic foci in the lungs. The successful completion of this project will increase our understanding of the mechanisms involved in development of distant metastases, and provide a base for development of anti-metastatic therapies needed for improvement of survival in patients with metastatic breast cancer.