Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated with inflammatory changes in bAT 2) to analyze for the first time the lipid profile of bAT in obese and lean mammary tumor-bearing and normal mice. Syngeneic E0771 mammary tumor cells were implanted into the mammary fat pad of lean and diet-induced obese C57BL/6 mice. BATs were analyzed four weeks after tumor cell inoculation by immunohistochemistry and mass spectrometry. Phospholipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan or neutral ion loss scan employing appropriate class specific lipid standards in a two step quantification process. Four main classes of phospholipids were analyzed: phosphatidylcholines phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols. Our results showed that bAT in obese (normal and tumor-bearing) mice contained hypertrophic adipocytes compared with their corresponding samples in lean mice; higher numbers of macrophages and crown-like structures were observed in obese tumor bearers compared to obese normal mice. BAT from normal obese mice revealed higher concentrations of phosphatidylethanolamines. Furthermore, bAT from tumor-bearing mice expressed higher phosphatidylcholines than that from non-tumor bearing mice, suggesting the presence of the tumor is associated with phosphatidylcholines. Conversion of phosphatidylethanolamines to phosphatidylcholines will be investigated in E0771 cells. Additional studies are projected to investigate macrophage activation by these specific classes of phospholipids. Occurrence of triglycerides and free fatty acids will be examined in bAT and similar lipidomic analyses will be carried out visceral adipose tissue, highly inflamed in obesity.

Obesity, an established risk factor for breast and other cancers, is associated with systemic inflammation and increased visceral adipose tissue. Adipose tissue is a normal constituent of the breast; however, the role of breast adipose tissue in breast cancer development, especially in the context of obesity, has not been addressed before. There is no information on the lipid composition of different fat depots in the body, especially in the context of obesity, and even less among obese tumor hosts. The study of the lipid composition of breast adipose tissue in diet-induced obese (DIO) tumor-bearing and normal mice and its impact in breast cancer progression is novel and has not been previously examined. New profiling methods employing shotgun lipidomics, a technique employed in mass spectrometric analysis using the direct loading of crude lipid extracts into an electrospray ionization source for intrasource separation and identification of numerous lipids, allow for extensive cellular lipid profiles of different tissues being accrued with relative ease. We studied the lipidomic profiles of the breast adipose tissue in lean and DIO normal and tumor bearing mice. Lipidomics analyses were performed using an electrospray triple quadrupole mass spectrometer (TSQ quantum Access Max) and class specific parent-ion or neutral loss scan in positive and negative ion mode with appropriate collision energy. The ratiometic quantification of lipids was done using class specific lipid standards. The phospholipid classes quantified were phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Our results for the PC class reveal an association between the total carbon chain of the lipids and the lipid concentrations based on four conditions: lean control, obese control, lean tumor bearers, and obese tumor bearers. The highest total carbon chain length is associated with the obese tumor condition. The next highest total carbon chain length is associated with lean tumor condition. This demonstrates that both the presence of the tumor as well as obesity play a role in contributing to a higher number of total carbons in the lipid chains. The other lipid classes analyzed express similar patterns from the data gathered when compared to the PC lipid class. Characterizing a particular lipid signature relevant to breast cancer and obesity may allow its targeting with therapeutic purposes. Citation Format: Osvaldo Perez, Michael Margolis, Ana M. Santander, Mitchell Martinez, Sanjoy Bhattacharya, Marta Torroella-Kouri. Breast cancer and obesity impact the lipid composition of breast adipose tissue: a preliminary study using shotgun lipidomics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3496. doi:10.1158/1538-7445.AM2014-3496

Most studies linking obesity and cancer focus on the systemic effects of adiposity in tumorigenesis. The mammary gland has white adipose tissue required for normal gland development, yet the plausible role that this local breast fat may play in breast cancer, especially in obese females, has been overlooked. Whether obesity also promotes breast cancer through its effect in local adipose tissue inflammation and innate immune signaling in the breast, where cancer occurs, has not been thoroughly investigated. Adipocytes and tumor cells in the breast may recruit macrophages to the tumor microenvironment contributing to tumor progression, particularly in obese females. We examined the interplay between these three cell types and its effects on macrophage chemotaxis in an in vitro setting, using co-cultures of mouse peritoneal macrophages, E0771 murine mammary tumor cells and in vitro differentiated or ex vivo isolated adipocytes from murine obese fat tissue. We also exposed macrophages in vitro to the individual or mixed paracrine factors leptin, lauric acid, estrogen and CCL2 produced by fat and mammary tumor cells to study macrophage chemotaxis, cellular differentiation and M1/M2 activation profiles. Specific signaling inhibitors of these paracrine factors were used to analyze reversion of these actions and proteomics analyses was undertaken to identify novel molecules secreted by adipocytes and mammary tumor cells with actions on macrophages or on the tumor microenvironment. We centered on the adipokine leptin, which has a main role in breast cancer progression, and we showed that leptin decreases pro-inflammatory IL-12, nitric oxide and VEGF production in macrophages but does not alter IL-10 production. A novel leptin-signaling inhibitor peptide was also used in in vivo experiments with diet-induced obese female C57BL6 mice bearing the syngeneic E0771 mammary tumor cells to analyze reduction of tumor progression, tumor-associated macrophage recruitment, crown-like structures in the breast adipose tissue and tumor angiogenesis. Our results underscore the relevance of the interactions between macrophages, adipocytes and tumor cells in the breast cancer microenvironment for tumor progression and demonstrate that the synergistic actions of various paracrine factors from these different cell types play the most crucial role in macrophage recruitment and tumor progression. Citation Format: Ana M. Santander, Tulay Koru-Sengul, Olivia Casas, Lidia Sanchez, Osvaldo Perez, Marta Torroella-Kouri. Paracrine interactions between macrophages, adipocytes and tumor cells in the breast cancer microenvironment of the obese female mouse contribute to tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1086. doi:10.1158/1538-7445.AM2014-1086

Breast cancer is the second leading cause of death by cancer in women in the United States. The occurrence of high numbers of macrophages in the tumor stroma has been associated with tumor progression and poor prognosis in breast and other solid malignances; however macrophage numbers have not been validated as a standard prognostic factor in the clinical practice. Breast adipose tissue is also part of the breast tumor microenvironment, and macrophages are observed in fat tissue surrounding dead adipocytes in “crown-like structures” (CLS). CLS have also been associated with poor survival in breast cancer; however they are not currently used in the assessment of breast cancer prognosis in the clinical practice either. This study was designed to determine whether the numbers of CD163+ tumor-associated macrophages (TAMs) and/or CD163+ macrophages in the breast adipose tissue (CLS) are independent prognostic factors in breast cancers across three different ethnic groups, African American, Latinas and Caucasian women. We also analyzed the polarization of macrophages as pro-inflammatory M1 (CD40+) and immunosuppressive M2 (CD206+) types, across these three ethnicities. A retrospective analysis of 150 breast cancer cases encompassing these three ethnic groups was carried out. African American and Latina women present with less incidence but more aggressive breast cancer disease and therefore, proportionally higher death rates. Using immunohistochemistry (IHC), we sought to identify whether there were any associations between the numbers of CD163+ TAMs and CLS with overall survival in these groups. Our findings show a statistically significant relationship between ethnicity and CD163+ macrophages located in the breast cancer microenvironment (TAMs/CLS) and reveal that the highest numbers of CD163+ TAMs/CLS are found in African American breast cancer patients. Latinas precede Caucasian breast cancer patients when assessing the numbers of CD163+ TAMs/CLS that were found, and Caucasian breast cancer patients show the lowest number of CD163+ TAMs/CLS. Using univariate survival analysis, our results show that the numbers of CD163+ TAMs and their M2 activation profile (CD206+) are associated with lower patient survival, whereas M1 (CD40+) macrophages are associated with higher patient survival. Univariate survival analysis also shows that the number of CLS exhibiting both M2/ M1 macrophages is associated with lower patient survival. We reveal for the first time that race/ethnicity is significantly associated with the number of TAMs and CLS in breast cancer. Citation Format: Ana M. Santander, Tulay Koru-Sengul, Feng Miao, Merce Jorda, Stephan Gluck, Consuelo Alvarez, Clara Milikowski, Osvaldo Perez, Mehrdad Nadji, Roberto Carrio, Omar Lopez-Ocejo, Marta Torroella-Kouri. Macrophages in the breast tumor microenvironment as a prognostic factor among race/ethnicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1286. doi:10.1158/1538-7445.AM2015-1286