Identifying the pro-metastatic mechanisms of neutrophil extracellular traps

项目摘要 每年，美国约有40，000名成功治疗原发性乳腺癌的妇女， 但仍有转移性复发。转移需要四个关键步骤：1）肿瘤细胞离开肿瘤; 2） 肿瘤细胞进入新的组织; 3）播散的肿瘤细胞（DTC）重新启动增殖;和4） 建立炎性微环境以支持生长的转移。步骤1-2经常发生 在癌症被诊断出来之前，很少进行干预，但我们可能能够针对步骤3-4 以减少转移的发生。为了实现这一长期目标，我们必须确定a）如何 静止的DTC重新启动增殖和B）支持转移的炎症微环境如何 是成立的。我们对中性粒细胞胞外陷阱（NETs）的研究提供了新的见解， 这些过程可能发生。NET由基因组DNA网格和大约40种相关蛋白组成， 由中性粒细胞释放到细胞外空间，以响应感染和炎症。小鼠 模型中，我们发现由烟草烟雾或细菌脂多糖诱导的肺部炎症， 静止的DTC重新启动增殖，导致致命的转移。通过活体成像，我们发现， 肺部炎症后，DTC被NET包围。我们发现NET上的蛋白酶 层粘连蛋白，一种基底膜蛋白。这种切割产生了激活整合素的层粘连蛋白表位 受体，触发静止DTC增殖。为了干扰这种机制，我们开发了一种 针对NET切割的层粘连蛋白（ChiAb 28）的抗体。ChiAb 28在超过一半的小鼠中阻止了转移， 减少了剩余小鼠的转移。因此，NET相关蛋白酶驱动转移性肿瘤的第3步， 过程同时，我们的新数据表明，其他NET相关蛋白激活巨噬细胞分泌 白细胞介素（IL）-1β，这反过来又诱导更多的NET和支持转移。此NET/巨噬细胞 反馈回路可产生支持转移的炎性微环境（步骤4）。 我们的数据使我们假设NET通过以下方式触发静止DTC的转移：a）裂解 层粘连蛋白重新启动增殖和B）驱动引起局部炎症的反馈环。NET如何 然而，切割层粘连蛋白仍然不清楚，我们将在目标1中确定这些机制。反馈是否经过 NET和巨噬细胞之间通过IL-1β分泌维持转移瘤生长的回路将是 在目标2中确定。最后，我们将联合收割机结合我们在NET生物学方面的独特专长，肿瘤 微环境和活体成像，以研究ChiAb 28和 化疗或IL-1β阻断抗体作为预防转移的新治疗方法。 本项目将确定负责NET推动转型能力的机制 从静止到增殖的DTC，并进一步到致死性转移。关于NET如何促进 转移可能最终允许开发NET靶向策略以预防转移。

PROJECT SUMMARY Every year, ~40,000 women in the US who had been successfully treated for primary breast cancer nonetheless have metastatic recurrence. Metastasis requires four key steps: 1) tumor cells leave the tumor; 2) tumor cells enter a new tissue; 3) disseminated tumor cells (DTCs) re-initiate proliferation; and 4) an inflammatory microenvironment is established to support the growing metastasis. Steps 1-2 often happen before the cancer is diagnosed and are rarely amenable to intervention, but we may be able to target steps 3-4 to reduce the occurrence of metastasis. To accomplish this long-term goal, we must determine a) how the quiescent DTCs re-initiate proliferation and b) how the metastasis-supporting inflammatory microenvironment is established. Our research on neutrophil extracellular traps (NETs) has provided novel insights into how these processes can occur. NETs consist of meshes of genomic DNA with ~40 associated proteins, and they are released by neutrophils to the extracellular space in response to infections and inflammation. In mouse models, we found that lung inflammation, induced by tobacco smoke or bacterial lipopolysaccharide, triggered quiescent DTCs to re-initiate proliferation, causing lethal metastases. Using intravital imaging, we found that DTCs were surrounded by NETs after lung inflammation. We discovered that proteases on NETs cleave laminin, a basement membrane protein. This cleavage generated a laminin epitope that activated integrin receptors, triggering quiescent DTCs to proliferate. To interfere with this mechanism, we developed an antibody against NET-cleaved laminin (ChiAb28). ChiAb28 prevented metastasis in over half of the mice and reduced metastasis in the remaining mice. Thus, NET-associated proteases drive step 3 of the metastatic process. In parallel, our new data show that other NET-associated proteins activate macrophages to secrete interleukin (IL)-1β, which in turn induces more NETs and supports the metastases. This NET/macrophage feedback loop may create an inflammatory microenvironment that supports the metastasis (step 4). Our data have led us to hypothesize that NETs trigger metastases from quiescent DTCs by a) cleaving laminin to re-initiate proliferation and b) driving a feedback loop that causes local inflammation. How NETs cleave laminin, however, is still unclear, and we will determine these mechanisms in Aim 1. How the feedback loop between NETs and macrophages via IL-1β secretion maintain the growth of the metastasis will be determined in Aim 2. Finally, we will combine our unique expertise in NET biology, the tumor microenvironment, and intravital imaging to investigate combination treatments with ChiAb28 and either chemotherapy or IL-1β blocking antibodies as new therapeutic approaches to prevent metastasis in Aim 3. This project will determine the mechanisms that are responsible for NETs' ability to drive the transition from quiescent to proliferating DTCs and further to lethal metastases. New knowledge of how NETs promote metastasis may ultimately allow the development of NET-targeting strategies to prevent metastasis.