Monocyte recruitment and macrophage regulation of tumor growth and angiogenesis

近期和长期职业目标：我的近期职业目标是进一步发展我在肿瘤免疫学方面的技能，主要是使用各种技术设计和进行自己的实验。此外，我的目标也是学会利用我的资源，包括设备和人员，以进一步了解单核细胞和巨噬细胞在肿瘤进展中的作用。我的长期职业目标是在一个学术机构获得教职，这使我有机会不仅继续进行基础研究，而且还可以将我的发现应用于家畜癌症临床试验。此外，我希望有能力教学生有关肿瘤免疫学，因为它涉及到兽医。关键要素研究职业发展计划：我的职业发展计划有多个关键要素。首先，也是最重要的，是我在道博士和亨森博士的指导下，他们在指导博士后研究员方面有相当丰富的经验。其次，CSU的动物癌症中心提供了大量的指导支持，并为合作，科学工作提供了开放的政策。该设施拥有现代化的设备，包括多台流式细胞仪，以及足够的空间供我进行研究。在动物癌症中心的程序是由许多研讨会和期刊俱乐部，我计划参加，并定期由教师和学生参加增强。这些是分享想法和形成合作的平台。最后，道博士和我与科罗拉多大学健康科学中心和国家犹太医学和研究中心的牢固关系允许进一步合作以及接触人类医学和免疫学研究的其他领域。这些研究所是免疫学研究的领先机构之一，因此我计划充分利用这一资源。项目概述：肿瘤相关巨噬细胞（TAM）在促进肿瘤生长中发挥重要作用。TAM被认为主要来自通过各种信号（即CCL 2和CSF-1）主动募集到肿瘤的单核细胞。然而，文献中的数据表明，TAM也可以来自中性粒细胞（PMN），使其成为一个有吸引力的人群进行检查。一旦进入肿瘤，单核细胞，可能还有中性粒细胞，分化成巨噬细胞，具有强效的免疫抑制以及肿瘤生长和血管生成促进特性。然而，目前缺乏对哪些骨髓源性细胞群体被招募到肿瘤以及肿瘤炎症在此过程中所起作用的良好理解。这种募集的动力学也不清楚，或者单核细胞/中性粒细胞分化为免疫抑制性TAM是否可逆。因此，假设肿瘤炎症是CCL 2/CSF-1产生以及单核细胞和PMN募集的主要驱动因素，并且通过抑制炎症、消耗单核细胞和/或PMN或激活新到达的巨噬细胞来中断这种募集可以显著抑制这些TAM的积累并抑制肿瘤生长。这些假设将通过三个具体目标来解决。目的1将评估肿瘤炎症对肿瘤CCL 2和CSF-1产生的影响以及这些因子在单核细胞/PMN募集和分化为TAM中的作用。这些实验将利用缺乏CCL 2或其受体的小鼠和缺乏生物活性CSF-1的小鼠来检查这些因子对单核细胞募集的作用。此外，当PMN募集被阻断时，将利用小鼠IL-8（KC）和IL-8受体敲除小鼠的抗体耗竭来检查肿瘤变化。在这些小鼠模型中，将进行微珠标记实验以跟踪肿瘤中单核细胞和PMN的流入和周转率。目的2探讨脂质体氯膦酸二钠或抗体介导的单核细胞和中性粒细胞耗竭对肿瘤组织中TAM蓄积和肿瘤生长的影响。目的3涉及使用先天免疫的有效激活剂来评估免疫刺激对单核细胞和PMN以及TAM激活和肿瘤内抑制性TAM群体的发展的影响。此外，这种先天性免疫刺激和脂质体氯膦酸盐消耗的组合将作为逆转TAM免疫抑制表型的新机制进行研究。这些研究将为TAM的生物学以及肿瘤如何调节其募集和分化提供重要见解。这些信息反过来将有助于设计治疗策略以克服肿瘤固有的免疫抑制环境。 公共卫生相关性（由申请人提供）：大多数肿瘤避免被人体免疫系统消除，即使它们矛盾地积累了大量称为巨噬细胞的免疫细胞。这些细胞在抑制免疫系统对肿瘤作出反应的能力方面发挥作用。我们将使用小鼠模型来了解为什么这些细胞在肿瘤中积累，以及去除这些细胞对肿瘤免疫的影响。

DESCRIPTION (provided by applicant): Immediate and Long term career goals: My immediate career goals are to further develop my skills in tumor immunology, mainly in designing and carrying out my own experiments using a wide variety of techniques. In addition, my goals are also to learn to utilize my resources available, both equipment and personnel in order to further our understanding of the role of monocytes and macrophages in tumor progression. My long term career goal is to obtain a faculty position at an academic institution which offers me the opportunity to not only continue conducting basic research but also allow for application of my findings to cancer clinical trials in domestic animals. In addition, I wish to have the ability to teach students about tumor immunology as it relates to veterinary medicine. Key elements research career development plan: There are multiple key elements to my career development plan. First, and foremost, is my mentoring under Drs. Dow and Henson, who combined have considerable experience in mentoring post-doctoral fellows. Secondly, the Animal Cancer Center at CSU provides plenty of mentoring support as well as an open door policy for collaborative, scientific work. The facility has modern equipment, including multiple flow cytometers, and plenty of space for me to conduct my research. The program at the Animal Cancer Center is enhanced by many seminars and journal clubs, which I plan to attend and are well-attended by faculty and students on a regular basis. These are platforms for which ideas are shared and collaborations formed. Lastly, Dr. Dow's and my strong relationship with the University of Colorado Health Sciences Center and National Jewish Medical and Research Center allows for even further collaboration as well as exposure to human medicine and other areas of immunological research. These institutes are among the leading institutions for immunology research, thus I plan to use this resource to its fullest potential. Project Summary: Tumor associated macrophages (TAM) play an essential role in promoting tumor growth. TAM are thought to arise primarily from monocytes that are actively recruited to the tumor by various signals (i.e. CCL2 and CSF-1). However, data available in the literature suggests that TAM can also arise from neutrophils (PMN), making them an attractive population to examine as well. Once in the tumor, monocytes, and possibly PMN, differentiate into macrophages with potent immunosuppressive as well as tumor growth and angiogenesis-promoting properties. However, at present there lacks both a good understanding of which populations of bone marrow derived cells are recruited to the tumors and the role that tumor inflammation plays in the process. Nor are the kinetics of this recruitment understood or whether the differentiation of monocytes/PMNs into immunosuppressive TAM is reversible. Therefore, it is hypothesized that tumor inflammation is a major driver of CCL2/CSF-1 production and monocyte and PMN recruitment, and that interrupting this recruitment by suppressing inflammation, depleting monocytes and/or PMN, or activating newly arrived macrophages can significantly suppress the accumulation of these TAM and inhibit tumor growth. These hypotheses will be addressed with 3 specific aims. Aim 1 will assess the effects of tumor inflammation on tumor CCL2 and CSF-1 production and the role of these factors in monocyte/PMN recruitment and differentiation into TAMs. These experiments will utilize mice lacking CCL2 or its receptor and mice deficient in biologically active CSF-1 to examine the role of these factors on monocyte recruitment. In addition, antibody depletion of mouse IL-8 (KC) and IL-8 receptor knockout mice will be utilized to examine tumor changes when PMN recruitment is blocked. Within these mouse models, microbead labeling experiments will be undertaken to track the influx and turnover rate of monocytes and PMN in tumors. Aim 2 addresses the effects of monocyte and PMN depletion-using liposomal clodronate or antibody-mediated PMN depletion on accumulation of TAM in the tumor and tumor growth. Aim 3 involves use of a potent activator of innate immunity to assess the effects of immune stimulation on monocytes and PMN as well as TAM activation and development of suppressive TAM population within tumors. In addition, the combination of this innate immune stimulus and liposomal clodronate depletion will be investigated as a novel mechanism of reversing the TAM immunosuppressive phenotype. These studies will provide important insights into the biology of TAM and how their recruitment and differentiation is regulated by tumors. This information will in turn be useful in designing therapeutic strategies to overcome the inherently immunosuppressive environment of tumors. PUBLIC HEALTH RELEVANCE (provided by applicant): Most tumors avoid elimination by the body's immune system, even though they paradoxically accumulate large numbers of immune cells known as macrophages. These cells play a role in suppressing the ability of the immune system to respond to the tumor. We will use mouse models to understand why these cells accumulate in tumors and the effects of removing these cells on tumor immunity.