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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Research Scientist Development Award - Research & Training (K01)
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National Center for Research Resources Initial Review Group (RIRG)
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Contreras, Miguel A
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Colorado State University-Fort Collins
Other Clinical Sciences
Schools of Veterinary Medicine
Fort Collins
United States
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Regan, Daniel; Guth, Amanda; Coy, Jonathan et al. (2016) Cancer immunotherapy in veterinary medicine: Current options and new developments. Vet J 207:20-28
Guth, Amanda M; Deogracias, Mike; Dow, Steven W (2014) Comparison of cancer stem cell antigen expression by tumor cell lines and by tumor biopsies from dogs with melanoma and osteosarcoma. Vet Immunol Immunopathol 161:132-40
Guth, A M; Hafeman, S D; Elmslie, R E et al. (2013) Liposomal clodronate treatment for tumour macrophage depletion in dogs with soft-tissue sarcoma. Vet Comp Oncol 11:296-305
Biller, B J; Guth, A; Burton, J H et al. (2010) Decreased ratio of CD8+ T cells to regulatory T cells associated with decreased survival in dogs with osteosarcoma. J Vet Intern Med 24:1118-23
Sottnik, Joseph L; Guth, Amanda M; Mitchell, Leah A et al. (2010) Minimally invasive assessment of tumor angiogenesis by fine needle aspiration and flow cytometry. Angiogenesis 13:251-8
U'Ren, Lance; Guth, Amanda; Kamstock, Debra et al. (2010) Type I interferons inhibit the generation of tumor-associated macrophages. Cancer Immunol Immunother 59:587-98