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，主要是利用各种技术设计和开展我自己的实验。此外，我的目标也是学会利用我现有的资源，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学术机构获得教职，不仅可以继续进行基础研究，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肿瘤免疫学，因为它与兽医学有关。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dr。Dow和Henson两人在指导博士后研究人员方面有着丰富的经验。其次，科罗拉多州立大学的动物癌症中心提供了大量的指导支持，并为合作的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，并有足够的空间供我进行研究。动物癌症中心的项目通过许多研讨会和期刊俱乐部得到加强，我计划参加这些活动，并且教师和学生经常参加这些活动。这些都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的合作，以及接触人类医学和其他免疫学研究领域。这些研究所是免疫学研究的主要机构之一，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（Tumor associated macrophages， TAM）在促进肿瘤生长中发挥着重要作用。TAM被认为主要是由通过各种信号（即CCL2和CSF-1）积极募集到肿瘤的单核细胞引起的。然而，文献中的数据表明，TAM也可以由中性粒细胞（PMN）引起，这使得它们也成为一个有吸引力的研究对象。一旦进入肿瘤，单核细胞，可能还有PMN，分化为巨噬细胞，具有有效的免疫抑制以及促进肿瘤生长和血管生成的特性。然而，目前对于哪些骨髓来源的细胞群被募集到肿瘤中以及肿瘤炎症在这一过程中所起的作用缺乏很好的了解。这种招募的动力学也不清楚，或者单核细胞/ pmn向免疫抑制性TAM的分化是否可逆。因此，我们假设肿瘤炎症是CCL2/CSF-1产生、单核细胞和PMN募集的主要驱动因素，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，抑制肿瘤生长。这些假设将有三个具体目标。目的1将评估肿瘤炎症对肿瘤CCL2和CSF-1产生的影响，以及这些因素在单核细胞/PMN募集和向tam分化中的作用。这些实验将利用缺乏CCL2或其受体的小鼠和缺乏生物活性CSF-1的小鼠来检测这些因素在单核细胞募集中的作用。此外，将利用小鼠IL-8 （KC）的抗体消耗和IL-8受体敲除小鼠来检测PMN募集被阻断时肿瘤的变化。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探讨单核细胞和PMN消耗（使用氯膦酸脂质体或抗体介导的PMN消耗）对肿瘤中TAM积累和肿瘤生长的影响。目的3涉及使用一种有效的先天免疫激活剂来评估免疫刺激对单核细胞和PMN的影响，以及肿瘤内TAM的激活和抑制性TAM群体的发展。此外，这种先天免疫刺激和氯膦酸脂质体消耗的结合将被研究作为逆转TAM免疫抑制表型的新机制。这些研究将为TAM的生物学以及它们的募集和分化如何受到肿瘤的调节提供重要的见解。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。

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.