Epicardial development is a process during which epithelial sheet movement, single cell migration and differentiation are coordinated to generate coronary arteries. Signaling cascades regulate the concurrent and complex nature of these three events. Through simple and highly reproducible assays, we identified small organic molecules that impact signaling pathways regulating these epicardial behaviors. Subsequent biochemical analyses confirmed the specificity of these reagents and revealed novel targets for the widely used Dorsomorphin (DM) and LDN-193189 molecules. Using these newly characterized reagents, we show the broad regulation of epicardial cell differentiation, sheet movement and single cell migration by Transforming Growth Factor β (TGFβ). With the DM analog, DMH1, a highly specific Bone Morphogenetic Protein (BMP) inhibitor, we demonstrate the cooperative yet exclusive role for BMP signaling in regulation of sheet migration. The action of DMH1 reveals that small organic molecules (SOM) can intervene on a single epicardial behavior while leaving other concurrent behaviors intact. All SOM data were confirmed by reciprocal experiments using growth factor addition and/or application of established non-SOM inhibitors. These compounds can be applied to cell lines or native proepicardial tissue. Taken together, these data establish the efficacy of chemical intervention for analysis of epicardial behaviors and provide novel reagents for analysis of epicardial development and repair.

Intake of saturated fat is a risk factor for ulcerative colitis (UC) and colon cancer. Changes in the microbiota have been implicated in the development of UC and colon cancer. The host and the microbiota generate metabolites that may contribute to or reflect disease pathogenesis. We used lipid class specific quantitative mass spectrometry to assess the phospholipid (PL) profile (phosphatidylcholine [PC], phosphatidylethanolamine [PE], phosphatidylinositol [PI], phosphatidylserine [PS]) of stool from mice fed a high fat (HFD) or control diet with or without induction of colitis-associated tumors using azoxymethane and dextran sodium sulfate. The microbiota was assessed using qPCR for several bacterial groups. Colitis-associated tumors were associated with reduced bulk PI and PE levels in control diet fed mice compared to untreated mice. Significant decreases in the relative quantities of several PC species were found in colitis-associated tumor bearing mice fed either diet. Statistical analysis of the PL profile revealed distinct clustering by treatment group. Partial least squares regression analysis found that the relative quantities of the PS class profile best predicted bacterial abundance of Clostridium leptum and Prevotella groups. Abundance of selected PL species correlated with bacterial group quantities. Thus, we have described that a HFD and colitis-associated tumors are associated with changes in phospholipids and may reflect host-microbial interactions and disease states.

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