Elizabeth A. Murphy, Ph.D. - US grants

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is the second leading cause of cancer death in the United States and the fourth most commonly diagnosed cancer. There is increasing recognition of diet-induced modulation of inflammatory responses as being central to the processes of human carcinogenesis. The argument for this stems from the observations that pro-inflammatory states are closely linked to tumor promotion. The consumption of the anti- inflammatory dietary constituents curcumin, which is a component of the Indian spice currie, and quercetin, a flavonoid present in various fruits and vegetables, have been associated with reduced CRC risk. Such components of diet are generally known to have a much wider safety margin with chronic use than are anti- inflammatory drugs, which make them more viable candidates to aid in cancer prevention. While a number of studies have examined the effects of dietary supplements on inflammation in cancer, none have specifically examined the role of tissue macrophages (Ms), primary mediators of inflammation, on these effects. Further, few have examined combinations of dietary supplements on the biological mechanisms of carcinogenesis. The overarching goal of this proposed project is to 1) determine the independent and combined effects of the anti-inflammatory dietary constituents curcumin and quercetin on CRC progression, and 2) to determine whether these benefits result from a reduction in M-induced inflammation. It is clear that M-induced inflammation plays an important part in the initiation and progression of CRC, and it may also be responsible for various sickness behaviors like fatigue, lack of appetite, and body wasting that can drastically decrease quality of life in CRC patients. A substantial proportion and perhaps the majority of CRCs are associated with non-genetic factors, such as inadequate and/or over nutrition, which can amplify inflammation in a number of ways. An independent, additive or synergistic effect of the anti-inflammatory dietary constituents curcumin and quercetin may be of critical public health significance in the prevention of CRC. The specific aims of this project are to 1) to elucidate the combined effects of curcumin and quercetin on M infiltration, inflammation, CRC progression and host survival and 2) to more specifically determine the role of M-induced inflammation on these effects. A mouse transgenic model of CRC will be used to determine the independent and combined effects of curcumin and quercetin on inflammatory processes, M infiltration, tumor progression and host survival at specific stages of CRC (e.g. prevention versus treatment). Furthermore, we will use M manipulation techniques to evaluate whether Ms are an important common pathway of the effects of curcumin and quercetin on inflammation, tumor progression and host survival. The overall goal of this project is to develop a clinically testable regimen to delay and/or prevent CRC and to begin to understand if the mechanisms of the effects are related to M infiltration and subsequent inflammation that could be targeted by further behavioral and or medical treatment. PUBLIC HEALTH RELEVANCE: The dietary compounds curcumin, a component of the Indian spice curry, and quercetin, a component of some fruits and vegetables, have anti-inflammatory properties and have been associated with reduced colon cancer risk. These compounds modify inflammatory processes which may be linked with the formation and development of tumors. The goal of the proposed study is to investigate the independent and combined effects of curcumin and quercetin on the progression of colon cancer and to determine if these effects result from a reduction in macrophage-induced inflammation.

ABSTRACT The development of the majority of breast cancers (BrCAs) is largely influenced by non-genetic factors such as high fat diet (HFD) induced obesity. The pathophysiological mechanisms that link HFD-induced obesity to BrCA risk include inflammatory processes; adipose tissue macrophages (M-Phi-s) are primary contributors to inflammation however there has been no systematic evaluation of their specific role in HFD-enhanced BrCA. Dietary compounds are of interest given their low toxicity profiles and their ability to target inflammation; however there is a fundamental gap in the understanding of their effectiveness and their mechanism(s) of action in HFD-enhanced BrCA. The long-term goal is to develop the anti-inflammatory flavonoid quercetin as a clinically testable dietary regimen to delay and/or prevent HFD-enhanced BrCA. The objective in this particular investigation is to evaluate whether M-Phi-s are a target for the anti-inflammatory effects of quercetin in HFD-enhanced BrCA and to determine if these effects are mediated through sirtuin 1 (SIRT1). The central hypothesis is that the mechanism of action of quercetin on the regulation of M-Phi-induced inflammation in HFD-enhanced BrCA is mediated through SIRTL This hypothesis has been formulated on the basis of several converging lines of evidence from the applicants' laboratory. The rationale for the proposed research is that elucidating the targets of quercetin and their mechanism of action in the regulation of these targets will translate to a more effective prevention/treatment approach in HFD-enhanced BrCA. This hypothesis will be tested by pursuing three specific aims: 1) Elucidate the stage-specific effects of quercetin on inflammation in HFD-enhanced BrCA; 2) Evaluate whether M-Phi-s are a target for the anti-inflammatory effects of quercetin in HFD-enhanced BrCA; and 3) Determine whether SIRT 1 is a mediator of the effects of quercetin in the regulation of M-Phi-induced inflammation in HFD-enhanced BrCA. Under the first aim, the C3(1)SV40Tag mouse model of BrCA, that will be fed a HFD to induce obesity, will be used to determine the stage-specific effects of quercetin on inflammation and subsequent tumorigenesis and overall survival. Under the second aim, in vivo M-Phi manipulation techniques will be used such as crossing the C3(1)Tag transgenic mouse model of BrCA with a MCP-1 knockout mouse to generate a M-Phi deficient mouse model of BrCA. This will help determine the role of M-Phi-s on the benefits of quercetin in HFD-enhanced BrCA. Finally, under aim 3, SIRT1 manipulation techniques will be used to examine the role of SIRT1 as a mediator of the effects of quercetin on M-Phi-induced inflammation in HFD-enhanced BrCA. The innovation of the proposed investigation is anchored in the examination of SIRT1 as a mediator of the benefits of the bioactive dietary component quercetin in the regulation of M-Phi-induced inflammation in HFD-enhanced BrCA. The proposed investigation is significant as it addresses prevention of incidence and progression of HFD-enhanced BrCA by using a dietary food component to target inflammation that is at the mechanistic core of this disease.

DESCRIPTION (provided by applicant): The development of the majority of colorectal cancers (CRCs) is largely influenced by non-genetic factors such as high fat diet (HFD)-induced obesity. The pathophysiological mechanisms that link obesity to CRC risk include inflammatory processes; adipose tissue macrophages (ATM¿s) are a primary source of inflammation, however, there has been no systematic evaluation of their specific role in CRC. There is evidence to confirm a regulatory role of miRNA-155 on M¿-induced inflammation; it has been shown that miRNA-155 directly inhibits suppressor of cytokine signaling 1 (SOCS1) in M¿s, thus increasing their inflammatory potential. While it is clear that miRNA-155 can positively regulate M¿-induced inflammation, there is a fundamental gap in the understanding of its role on the regulation of ATM¿-induced inflammation in HFD enhanced CRC. The long- term goal is to understand the role of ATM¿s in HFD enhanced CRC that could be targeted using behavioral and or medical treatments. The objective of this particular investigation is to determine the role of ATM¿s on inflammation and subsequent progression of HFD enhanced CRC, and further, to evaluate if this process is regulated by miRNA-155. The central hypothesis is that the regulation of ATM¿-induced inflammation in HFD- enhanced CRC is mediated through miRNA-155. The rationale for the proposed research is that elucidating the link between obesity and CRC will translate to a more effective prevention/treatment approach. This hypothesis will be tested by pursuing two specific aims: 1) Determine the role of ATM¿s on inflammation and subsequent progression of CRC; and 2) Evaluate the role of miRNA-155 on regulation of ATM¿-induced inflammation in CRC. Under the first aim we will compare the effects of three HFDs (40% of total calories) differing in the percentage of saturated fat (6%, 12% & 24% of total calories) on CRC progression in the AOM/DSS mouse model of CRC, and further, examine the association between CRC, adipose tissue inflammation, M¿ polarization, and expression of miRNA-155. Further, using adoptive transfer of ATM¿s from mice fed HFDs to CRC mice fed normal diets (ND) we will directly determine the role of ATM¿s on progression of CRC. In the second aim, we will test the hypothesis that miRNA-155 plays a critical role in the regulation of M¿-induced inflammation in HFD enhanced CRC. To this end, using a miRNA-155-/- mouse and AOM/DSS to initiate CRC we will examine the role of miRNA-155 on ATM¿-induced inflammation and subsequent tumorigenesis. Further, using adoptive transfer of ATM¿s from HFD miRNA-155-/- mice to wildtype CRC mice that will be fed a ND we will determine if the effects of ATM¿s on CRC progression are mediated through miRNA-155. The innovation of this investigation is anchored in the examination of the role of ATM¿s in CRC, and further, the regulation of these M¿s by miRNA-155. The proposed investigation is significant as it addresses prevention of incidence and progression of HFD-enhanced CRC. If this hypothesis is correct miRNA-155 may be an important therapeutic target of ATM¿-induced inflammation in HFD-enhanced CRC.

DESCRIPTION (provided by applicant): Dr. Angela Murphy joined the Department of Pathology, Microbiology and Immunology at the University of South Carolina as an Assistant Professor in September 2010. She is committed to a health- related research career as evident by her 40 peer-reviewed journal articles in the area of complementary and alternative (CAM) treatment approaches to infection, inflammation and cancer. Her long-term career goal is to significantly contribute to expanding global knowledge in CAM treatments for cancer. This will largely involve sustaining an independent line of translational research, engaging in cutting edge techniques, generating R01 funding, publishing in high impact journals, presentations at meetings, training students and junior faculty, technology transfer and drug discovery, as well as involvement in program projects and center grants. Her short-term goals are to expand her research and training experiences providing her with the tools to generate an independent research program and an R01 grant before the end of this proposed career development award. In the proposed investigation, Dr. Murphy will examine the role of miRNA-155 on the regulation of macrophage behavior in a mouse model of obesity-enhanced CRC, and further, whether dietary quercetin can target this process. While it is clear that she is interested in pursuing a career in CAM treatments for cancer, her current training is in Exercise Physiology. Therefore, the proposed studies will provide her with new training and expertise that she needs to become a leading immunologist with the tools to perform mechanistic and translational research on CAM in cancer. Overall, these studies will allow Dr. Murphy to gain the experience and expertise in new areas and using new techniques that will greatly enhance her ability to generate new funding, and thus, have a significant impact on the prevention of colorectal cancer. The University of South Carolina is an outstanding environment for the career development of Dr. Murphy in both successful mentors and the research resources that are available. Dr. Murphy's mentors and Advisory Committee members have a strong record of mentoring junior faculty in both basic science and clinical research in the area of CAM, inflammation and cancer. Further, the institutional research infrastructure that is available to Dr. Murphy is exceptional and will allow for successful completion of the proposed investigation. The proposed career development plan was carefully devised by Dr. Murphy and her mentors and is tailored to her ability. It focuses on five major aspects of training that she is in most need of including, 1) Meetings with Mentors and Advisory Committee Members; 2) Research Training; 3) Grant Writing; 4) Courses; and 5) Manuscript Writing. Dr. Murphy will also participate in Working Groups; Weekly Lab Meetings; Journal Club; Seminars; Workshops; and Conferences as part of the training plan. This training plan, her mentors and the research infrastructure will provide her with the necessary skills and expertise to become a highly competent independent investigator performing multidisciplinary research on CAM in cancer. A pathophysiological mechanism that may link obesity to colorectal (CRC) risk is inflammation. Adipose tissue macrophages are a primary source of inflammation; however, there has been no systematic evaluation of their regulation in obesity-enhanced CRC. miRNA-155 inhibits signaling pathways in macrophages that can suppress inflammation. It is upregulated during the macrophage inflammatory response and has been implicated in playing a role in the link between inflammation and cancer. However, there are no reports of a role of miRNA-155 in obesity-enhanced CRC. Dietary compounds are of interest given their low toxicity profiles and their ability to target inflammatio; however, there is a fundamental gap in the understanding of their effectiveness and their mechanism(s) of action. The long-term goal is to develop the flavonoid quercetin as a preventative/therapeutic strategy for obesity-enhanced CRC. The objective of this investigation is to evaluate whether macrophage-induced inflammation is regulated by miRNA-155 in obesity-enhanced CRC, and whether dietary quercetin can target this process. Further, we will begin to evaluate the clinical efficacy of miRNA-155 as a biomarker for CRC. The central hypothesis is that regulation of macrophage-induced inflammation in obesity-enhanced CRC is mediated through miRNA-155, a process that can be targeted by dietary quercetin. The rationale is that elucidating the targets of quercetin and their mechanism of action will translate to a more effective prevention/treatment approach in obesity-enhanced CRC. This hypothesis will be tested under three Specific Aims: 1) Determine the role of miRNA-155 in the regulation of macrophage-induced inflammation in obesity-enhanced CRC; 2) Evaluate whether miRNA-155 can be targeted by dietary quercetin; and 3) Determine the clinical efficacy of miRNA-155 as a biomarker for CRC. In Aim 1, we will use a miRNA-155 -/- mouse in which obesity will be induced by high fat diet (HFD) and CRC induced using AOM/DSS. We will examine inflammation and macrophage behavior in adipose tissue, immune regulation in the tumor microenvironment, and tumorigeneis. In Aim 2, we will test the hypothesis that quercetin can decrease expression of miRNA-155 in macrophages, and thus, decrease tumorigenesis in obesity-enhanced CRC. In Aim 3, we will employ a case-control design to being to determine the clinical efficacy of miRNA-155 as a biomarker for CRC, and further, assess the associations and predictive ability of miRNA-155 and advanced-stage and high-grade. The proposed investigation is significant as it addresses prevention of incidence and progression of obesity-enhanced CRC by using a dietary food component to target macrophage-induced inflammation that is thought to at the mechanistic core of this disease.

DESCRIPTION (provided by applicant): High-fat diet (HFD)-induced obesity increases the risk for colorectal cancer (CRC). A pathophysiological mechanism that may link obesity to CRC risk is inflammation. Adipose tissue macrophages (ATM¿s) are a primary source of inflammation; however, there has been no systematic evaluation of their regulation in HFD-enhanced CRC. miRNA-155 (miR-155) inhibits signaling pathways in M¿s that can suppress inflammation. It is upregulated during the M¿ inflammatory response and has been implicated in playing a role in the link between inflammation and cancer. However, there are no reports of a role of miR-155 in HFD-enhanced CRC. Dietary compounds are of interest given their low toxicity profiles and their ability to target inflammation; however, there is a fundamental gap in the understanding of their effectiveness and their mechanism(s) of action. The long-term goal is to develop the flavonoid quercetin as a preventative/therapeutic strategy for obesity-enhanced CRC. The objective of this investigation is to evaluate whether M¿-induced inflammation is regulated by miR-155 in obesity-enhanced CRC, and whether dietary quercetin can target this process. The central hypothesis is that regulation of M¿-induced inflammation in obesity-enhanced CRC is mediated through miR-155, which may be an important mediator of quercetin action. The rationale is that elucidating the molecular links between obesity and CRC and identifying strategies to target these actions will translate to a more effective prevention/treatment approach in HFD-enhanced CRC. This hypothesis will be tested under two specific aims: 1) Determine the role of miR-155 in the regulation of M¿-induced inflammation in HFD-enhanced CRC; 2) Evaluate whether miR-155 can be targeted by dietary quercetin in HFD-enhanced CRC. In aim 1, we will use a miR-155-/- mouse in which obesity will be induced by HFD and CRC will be induced using AOM/DSS. We will examine inflammation and M¿ behavior in adipose tissue, immune regulation and inflammation in the tumor microenvironment, as well as tumorigenesis. Further, adoptive transfer of ATM¿s from both HFD wildtype and HFD miR-155-/- donor mice to wildtype recipient mice will be performed to determine if the effects of HFD on CRC are directly mediated through ATM¿s, and moreover, if this process is regulated by miR-155. In aim 2, we will determine if quercetin feedings can decrease expression of miR-155 in ATM¿s and if this is associated with a decrease in M¿-induced inflammation and reduced tumorigenesis. Further, using miR-155-/- mice we will determine if quercetin is mediating its effects through this miRNA. We will use adoptive transfer of ATM¿s from WT and miR-155-/- mice fed quercetin to directly determine if the benefits of quercetin on inflammation in HFD-enhanced CRC are mediated through ATM¿s. The proposed investigation is significant as it addresses prevention of incidence and progression of obesity-enhanced CRC by using a dietary food component to target M¿-induced inflammation, which is thought to at the mechanistic core of this disease.

? DESCRIPTION (provided by applicant): Emerging evidence links gut microbiota to the development and progression of colon cancer (CoCA). Interestingly, obesity is a significant driver for the composition of gut microbiota. However, there are no studies that have directly examined the influence of gut microbiota on driving obesity-enhanced CoCA. The tumor- promoting effects of the microbiota in CoCA are caused, at least in part, by altered host-microbial interactions. Host-derived immune and inflammatory responses are important driving forces that shape microbial community composition and, when altered, may contribute to dysbiosis. Macrophages (M?s) are a primary source of obesity-associated inflammation and have been linked to poor prognosis in CoCA. However, there are no studies that have systematically examined a role for M?s in altering the gut microbial profile in obesity- enhanced CoCA. The long-term goal is to uncover the mechanisms linking obesity to CoCA. The objective in this particular investigation is to determine the influence of an obesity-induced alteration in ut microbiota on CoCA progression and to test whether this process is regulated by M?s. The central hypothesis is that obesity-induced changes in gut microbiota are driven by M?-mediated inflammatory processes and lead to enhanced progression of CoCA. This hypothesis will be tested by pursuing two specific aims: 1) Determine the effects of an obesity-induced alteration in gut microbiota on CoCA; and 2) Evaluate the role of M?s in altering the gut microbial profile in obesity-enhanced CoCA. Under the first aim, we will test the hypothesis that obesity can enhance CoCA by altering gut microbiota. Using an inducible genetic mouse model of colon cancer, we will examine the effects of diet-induced obesity on the gut microbial profile and subsequent progression of tumorigenesis. Further, we will transfer gut microbes from obese mice to lean mice carrying an inducible Apc mutation to test the influence of an obesity-induced alteration of gut microbiota on CoCA progression. Finally, using antibiotics we will deplete gut microflora from obese mice to further confirm the role of obesity-induced alterations in gut microbiota on CoCA. In the second aim, we will test the hypothesis that M?s play a necessary role in altering gut microbiota in obesity-enhanced CoCA. Using M? depletion techniques, we will examine the role of M?s in altering the gut microbial profile and subsequently promoting progression of tumorigenesis in a mouse model of obesity-enhanced CoCA. Further, using adoptive transfer of M?s from obese mice to lean mice carrying an inducible Apc mutation, we will directly determine their effects on gut microbiota and CoCA progression. The proposed investigation is significant as it will uncover a mechanism that links obesity to CoCA. Understanding the impact of obesity-induced alterations in gut bacteria on CoCA will allow us to translate to the development of approaches that will identify individuals at risk for CoCA. This would present enormous potential for prevention of CoCA in obese individuals through development of personalized dietary intervention strategies designed to restore a more healthy microbial profile in the gut.

Summary: Breast cancer is the most prevalent cancer and the second leading cause of cancer-related death in women in the United States. However, little progress has been made in preventing the incidence of breast cancer. Most breast cancer mortality results from metastatic recurrence after initial success of surgery and/or other therapies. Although extended treatment with selective estrogen receptor modulators and aromatase inhibitors have been proven effective in preventing metastatic recurrence of breast cancer, the benefit is limited to ER+ breast cancer, and severe adverse effects make them suboptimal for long-term use. Currently there are no preventive agents for ER- breast cancer. This is of particular relevance given the high metastatic recurrence rate and the resulting poor prognosis of triple negative breast cancer. One common feature of breast cancer, regardless of the subtypes, is the pro-tumor nature of the tumor microenvironment, which contains abundant M?s, called tumor-associated macrophages (TAMs). TAMs promote tumor development and metastasis through inducing immunosuppression, promoting angiogenesis, enhancing tumor cell proliferation and invasiveness, and facilitating cancer stem cell (CSC) formation and maintenance. Due to their determinant roles in all stages of cancer development, TAMs have been considered as a therapeutic target for cancer. However, M?s have not yet been exploited as a target for breast cancer prevention. Emodin is a small molecule compound derived from many plants including several Chinese herbs. Our published studies showed that emodin inhibited breast cancer growth and metastasis in orthotopic mouse models through reducing M? recruitment to tumors and lungs and suppressing their M2-like polarization by acting on both M?s and breast cancer cells. Our preliminary studies further showed that emodin could suppress TGF?1-induced epithelial to mesenchymal transition and diminish the stemness of breast cancer cells, and reduce the death due to metastatic recurrence after surgical removal of primary tumors in an orthotopic breast cancer model. Importantly, a comprehensive NIH toxicology study showed that emodin is very safe for long-term use in mice and rats. Given the important roles of M?s in breast cancer initiation and metastatic recurrence, and based on our published and preliminary data, we hypothesize that emodin can be developed as a safe and effective chemopreventive agent for breast cancer incidence and metastatic recurrence by virtue of its ability to block the tumor-promoting crosstalk between cancer cells and M?s. Two specific aims are proposed: SA1. To determine the effects of emodin on preventing breast cancer onset and post-surgery metastatic recurrence in mouse models; and SA2. To elucidate the mechanisms by which emodin prevents breast cancer onset and metastatic recurrence. The success of the proposed preclinical studies will set a stage for clinical trials to develop emodin as a new safe, effective and low-cost chemopreventive agent for breast cancer regardless of the subtype.

PROJECT SUMMARY Affecting upwards of 4 million people in North America and Europe, with an economic burden of $30 - $45 billion, Inflammatory Bowel Diseases (IBDs) are debilitating, significantly affect life-style, and carry a high colon cancer risk. Because conventional treatment outcomes are modest with dangerous side effects, about half of IBD patients turn to complementary and alternative medicines (CAMs). Although CAMs have been used for thousands of years, there is a gap in our knowledge of the mechanisms supporting their effectiveness. Understanding these mechanisms will lead to standardized treatment for IBD outside of toxic FDA-approved drugs. This will lower their colon cancer risk. Over the past decade, we have shown that American Ginseng (AG) suppresses colitis and prevents colon cancer in mice. Using scientifically rigorous Bioassay-Guided Fractionation, we have isolated a polyacetylene called panaxynol (PA) that has anti-inflammatory and anti-cancer properties. PA (compared to the100's of other CAMs being tested) comes from a natural source, and is a single ingredient, allowing it to be standardized on its own, or in a cocktail. What makes this molecule particularly interesting and innovative is the mechanism - it is a single molecule extracted from AG, with a unique capacity to target macrophages (m?) for apoptosis. Our long-term goal is to identify the primary component(s) of AG responsible for the robust anti-inflammatory and chemopreventive properties of AG we have observed over the past decade; and to determine their mechanism of action. The overall objective of this application is to gain a deeper understanding of both: (a) the broad treatment potential of PA (i.e. multiple pharmacologic and bioengineered animal models of colitis and colon cancer); and (b) the underlying mechanism(s) behind the observation that PA targets m? for apoptosis. We focus here on a DNA-damage independent p53 signaling pathway as a mechanism toward m? apoptosis. The scientific premise underlying the proposed research is robust. Comparing nine FDA-approved drugs, small molecules, and CAMs, PA is the most efficacious at suppressing colitis in a DSS mouse model. Our central hypothesis is that PA, isolated after a decade of rigorous bioassay-guided fractionation, has anti-inflammatory and anti-cancer activity in the colon because it activates p53- mediated apoptosis in infiltrating m?; mitigating colitis; and preventing colon cancer associated with colitis. Furthermore, PA acts as an anti-inflammatory in these models because it induces p53 through a DNA damage- like signaling response in m? that is independent of detectable DNA damage. To address this hypothesis, we will test the efficacy of PA in three mouse models of colitis and in genetically engineered mice. Because it appears that PA is taking advantage of a unique p53 mechanism in m?, we will test PA in mice with p53 conditionally knocked out in colonic m?. A DNA damage-independent mechanism is explored. Results consistent with our hypothesis would identify an innovative, low cost, safe, specific, and natural compound with anti-inflammatory and cancer chemopreventive properties that could quickly be implemented clinically.

PROJECT SUMMARY It has long been suspected that tumor resection surgery may actually accelerate cancer metastasis in some patients. Two theories for this have been proposed. The first one hypothesizes that some cancer cells are squeezed out of the tumor into circulation during the surgical operation and travel to distant organs to form metastasis, which grow into detectable metastasis several months later. The other hypothesizes that subclinical micrometastases have already seeded in distant organs even before the surgery but are under immune control, a state called metastatic dormancy, and the surgery somehow may accelerate the growth of the micrometastases into macrometastases by breaking the dormancy. Although clinical evidences strongly favor the second scenario, it is impractical and unethical to perform pseudo-surgery to directly test this hypothesis in patients. A very recent elegant animal study in Dr. Robert Weinberg?s laboratory strongly supports this scenario. Using a unique immunogenic syngeneic breast cancer mouse model, they demonstrated that surgery wounding results in systemic inflammation, during which the inflammatory monocytes and their resulting pro-tumor M2 macrophages (M?s) are mobilized into circulation, leading to accumulation of tumor-promoting M?s in the distant organs where they facilitate the metastasis establishment by the pre-surgery seeded of tumor cells. More interestingly, perioperative administration of meloxicam, a nonsteroidal anti-inflammatory drug (NSAID), could significantly suppress post-surgery tumor outgrowth, which is in line with a long time clinical observation that anti-inflammatory analgesia reduces post-surgery breast cancer relapse. Because NSAIDs may cause immunosuppression, wound healing delay, and other severe side effects, safer anti-inflammatory drugs are needed for this clinical application. In the past few years, our labs discovered that a Chinese herb-derived small molecule compound, emodin, has context-dependent bi- directional effects on M? activation, and can inhibit breast cancer growth and metastasis by reducing recruitment and M2-like polarization of tumor-associated M?s. We propose that emodin can be developed as a safe, low-cost, and effective complementary agent to be used perioperatively to alleviate the surgery triggered systemic inflammatory response and reduce resulting metastatic relapse of breast cancer. To test this hypothesis, we recently developed a 4T1-derived cell line, 4T1-Luc2-RFP. Orthotopic tumors formed by 4T1-Luc2-RFP cells display much slower growth and significant resistance to metastasis. This new cell line will present a good model with an appropriate time window to study the impact of surgery on tumor metastasis and to develop therapeutic strategies. Two aims are proposed: 1) to test if emodin can inhibit surgical wounding accelerated breast cancer growth and metastasis, and 2) to determine the role of macrophages in emodin?s actions on breast cancer metastasis.