A platform to functionally sort and analyze tumor cells within combinatorial metastatic micorenvironments

转移是造成约90%的人类癌症相关死亡的原因，但我们对转移阶段的理解， 转移和驱动继发性、第三性等肿瘤的调节特征严重缺乏。特别是， 散布细胞周围的早期生态位对于存活、休眠和/或成功的 发生进展性微转移。事实上，并非罕见，乳腺癌患者屈服于 治疗后数年至数十年复发或转移性疾病，使疾病无法检测。 事实上，超过67%的乳腺癌死亡发生在5年生存窗口期之后，一些患者 在十多年“无病”后复发。然而，我们对 启动和维持休眠与转移的程序的内在和环境因素 进展仍然极为有限。在这里，我们试图阐明基本的物理和分子 控制异位部位细胞命运的机制。迄今为止，许多技术障碍阻碍了我们的 研究休眠和复发的遗传和微环境驱动因素的能力，特别是在体内 这些事件很罕见，不容易控制事实上，允许控制细胞的体外平台 微环境和允许基于细胞状态（即休眠与进展）的细胞分离， 识别和表征管理这些行为的分子机制，这些机制可以被验证和针对 in vivo.为了应对这些重大挑战，该提案利用了癌症生物学和癌症方面的专业知识， 通过许多创新技术进行生物工程（例如微流体产生转移性小生境， 先进的光学成像，尖端的细胞工程与CRISPR技术等）这使得 这将极大地提高我们对休眠在体内是如何调节的理解。在这里，我们假设， 转移性小生境中的休眠或集落增殖由癌细胞之间的锁和钥匙行为决定 与特定的遗传和表观遗传信号和异位的初始和进化特性， 微环境我们的假设将在以下具体目标进行检验：（1）定义具体的 细胞外基质组合物，其使用高通量微生物技术驱动存活、休眠或定殖， 工程转移环境（MEME）技术;（2）解剖分子机制， 存活、休眠或在确定的转移性小生境微环境中定殖;（3）确定特定的 骨髓源性和组织特异性驻留巨噬细胞对癌细胞存活影响， 休眠或殖民通过这些努力，我们将剖析扩散性肿瘤的机制驱动因素， 细胞休眠或增殖，这将阐明治疗靶点，以防止休眠的肿瘤细胞， 逃避治疗此外，这些研究将揭示直接杀死休眠细胞或 防止它们从休眠状态逃逸到增殖状态，以防止复发。

Metastases are responsible for ~90% of human cancer-related deaths, yet our understanding of the stages of metastasis and the regulating features that drive secondary, tertiary etc. tumors is sorely lacking. In particular, the early niche surrounding disseminated cells appears critical for survival, dormancy, and/or successful development of progressing micrometastases. Indeed, not infrequently, breast cancer patients succumb to recurrent or metastatic disease years to decades after treatment that had rendered the disease undetectable. In fact, greater than 67% of breast cancer deaths occur beyond the 5-year survival window and some patients present with recurrence after more than a decade of being “disease-free”. Yet, our understanding of the intrinsic and environmental factors that initiate and maintain programs of dormancy versus metastatic progression remains extremely limited. Here, we seek to elucidate fundamental physical and molecular mechanisms that govern cell fate in ectopic sites. To date, numerous technical hurdles have impeded our ability to study the genetic and microenvironmental drivers of dormancy and recurrence, particularly in vivo where these events are rare and not easily controlled. Indeed, in vitro platforms that permit control of the cell microenvironment and permit cell isolation based on cell state (i.e. dormant vs. progressing) are required to identify and characterize molecular mechanisms governing these behaviors that can be validated and targeted in vivo. To address these significant challenges, this proposal leverages expertise in cancer biology and cancer bioengineering through numerous innovative technologies (e.g. microfluidic generation of metastatic niches, advanced optical imaging, cutting edge cell engineering with CRISPR technologies etc.) that uniquely enable us to drastically improve our understanding of how dormancy is regulated in vivo. Here, we hypothesize that dormancy or colony proliferation in metastatic niches is dictated by lock-and-key behavior between cancer cells with specific genetic and epigenetic signaling and the initial and evolving properties of the ectopic microenvironment. Our hypotheses will be tested in the following Specific Aims: (1) Define specific extracellular matrix compositions that drive survival, dormancy, or colonization using high-throughput micro- engineering metastatic environments (MEME) technology; (2) Dissect the molecular mechanisms governing survival, dormancy, or colonization in defined metastatic niche microenvironments; (3) Define the specific influence of bone marrow-derived and tissue-specific resident macrophages in carcinoma cell survival, dormancy, or colonization. Through these efforts we will dissect the mechanistic drivers of disseminated tumor cell dormancy or proliferation, which will elucidate therapeutic targets to prevent dormant tumor cells from evading therapy. Additionally, these studies will reveal therapeutic targets to kill dormant cells directly or prevent their escape from dormancy to proliferation in order to prevent recurrence.