Identifying the pro-metastatic mechanisms of neutrophil extracellular traps

项目总结 每年，美国约有4万名成功治疗原发性乳腺癌的女性 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)播散性肿瘤细胞(DTC)重新启动增殖；以及4) 炎性微环境的建立为肿瘤的生长提供了支持。步骤1-2经常发生 在癌症被诊断之前，很少接受干预，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DTCs重新启动增殖和b)支持转移的炎性微环境如何 已经确立了。我们对中性粒细胞胞外陷阱(Net)的研究为我们提供了新的见解 这些过程可能会发生。Net由基因组DNA和约40个相关蛋白质组成，它们 由中性粒细胞释放到细胞外空间，以应对感染和炎症。在鼠标中 模型，我们发现由烟草烟雾或细菌脂多糖引起的肺部炎症 静止的树突状细胞重新启动增殖，导致致命转移。使用活体成像技术，我们发现 DCs在肺部炎症后被网状物包围。我们发现网状物上的蛋白水解酶 层粘连蛋白，一种基底膜蛋白。这种切割产生了一个层粘连蛋白表位，激活了整合素 受体，触发静止的DTCs增殖。为了干扰这一机制，我们开发了一种 抗网状裂解层粘连蛋白抗体(ChiAb28)。ChiAb28阻止了超过一半的小鼠的转移， 减少其余小鼠的转移。因此，网络相关的蛋白水解酶驱动转移的第三步。 进程。与此同时，我们的新数据显示，其他网络相关蛋白激活巨噬细胞分泌 白介素1β，进而诱导更多的Net并支持转移。这个网状/巨噬细胞 反馈环可能会创造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a)裂解从静止的DTC触发转移 层粘连蛋白重新启动增殖，以及b)驱动引起局部炎症的反馈循环。How Nets 然而，裂解层粘连蛋白仍然不清楚，我们将在目标1中确定这些机制。 Net与巨噬细胞之间的环路通过IL-1β的分泌维持肿瘤的生长转移 最后，我们将结合我们在网络生物学方面的独特专业知识，肿瘤 微环境和活体成像，以研究ChiAb28和 化疗或IL-1β阻断抗体作为预防转移的新治疗方法在AIM 3中。 该项目将确定负责NETS推动过渡的能力的机制 从静止期到增殖期，再到致命的转移。关于篮网如何推广的新知识 转移可能最终允许发展网络靶向策略来防止转移。

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.