OBJECTIVES/GOALS: In triple negative breast cancer (TNBC), obesity is associated with poor outcomes. Adipose stem cells (ASCs) from obese patients (obASCs) secrete higher levels of adipokines compared to ASCs from lean individuals. Leptin, one of these adipokines, has been implicated in many cancers. This study seeks to examine the role of leptin signaling in TNBC. METHODS/STUDY POPULATION: Previous work in conjunction with a collaborating lab has shown that leptin signaling promotes metastasis and increased expression of epithelial-mesenchymal transition (EMT) markers in triple negative breast cancer cell lines. This project expands upon this work through using both patient-derived cell lines and and patient-derived xenografts (PDX), and examines the role of leptin signaling both in vitro and in vivo. To determine the effects of obesity upon a PDX model of TNBC, a high fat diet was used to induce obesity in vivo. A pharmacological inhibitor of the leptin receptor was used to test the requirement for leptin signaling both in vivo and in vitro. RESULTS/ANTICIPATED RESULTS: Exposure to conditioned media harvested from obASCs increased the percentage of TNBC cells that expressed cancer stem cell markers, whereas exposure to an inhibitor of the leptin receptor decreased the percentage of cells with cancer stem cell markers. PDX tumors implanted into mice with diet-induced obesity had an increased volume compared to tumors implanted into lean controls. Further analysis will be conducted on metastasis, circulating tumor cells, and survival in both lean and obese mice. DISCUSSION/SIGNIFICANCE: Understanding the complex signaling events in the obese tumor microenvironment is essential, as these molecular differences may contribute to different outcomes for obese and lean individuals with triple negative breast cancer. Therefore, study of the crosstalk between obASCs and TNBC cells is critical.

OBJECTIVES/GOALS: Current approaches to drug development for the aggressive triple negative breast cancer rely on current 2D and 3D in vitro models which have limited capabilities. We have developed a translational microphysiological system that can maintain the human breast microenvironment to capture the complex interaction with the tumor microenvironment. METHODS/STUDY POPULATION: Three different TNBC cell lines were seeded in BC-MPS: MDA-MB-231 parental cell line, MDA-MB-231wiht the gene, LKB1 overexpressed, which is a tumor suppressor, and MDA-MB-231 with the enzyme, ERK5, an enzyme associated with increased metastasis and drug resistance, knocked out. These three TNBC cell lines were cultured in a standard 2D 96-well plate and in BC-MPS. Time-lapse videos were taken to track cellular mobility. RNA-sequencing was performed to compare different expression levels of various cancer related genes of the cell lines cultured in standard 2D and BC-MPS. RESULTS/ANTICIPATED RESULTS: The LKB1 overexpressed MDA-MB-231 and the ERK5-ko MDA-MB-231 cell lines are expected to have decreased mobility compared to the parental cells. The cell lines are expected to have increased expression of cancer related genes when cultured in BC-MPS than when cultured in standard 2D due to the presence of human breast tissue. DISCUSSION/SIGNIFICANCE: BC-MPS is a promising new translational MPS that facilitates studying long term interactions between real human breast tissue and cancer cells. The BC-MPS systems ability to support the growth of established cell lines has been demonstrated. Future studies will focus on developing the model for personalized medicine.

ABSTRACT IMPACT: Identifying an important pathway in treatment resistant TNBC will allow for the future development of clinical therapeutics specific for this disease. OBJECTIVES/GOALS: Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer characterized by negative expression of estrogen receptor, progesterone receptor, and HER2/neu amplification. It resists therapies and has a high recurrence rate after resection. The goal of my research is to identify & characterize a TNBC pathway for future development of therapies. METHODS/STUDY POPULATION: The project uses a combination of cell lines, patient derived xenograft (PDX) models, as well as patient databases. Standard cellular and molecular biology techniques will be used including: Cell culture, qPCR, western blotting, and flow cytometry. RESULTS/ANTICIPATED RESULTS: LKB1 is a master kinase that activates 14 possible downstream kinases. The signaling pathway has been demonstrated to play a role in energy homeostasis and metabolism. Mutation of LKB1 signaling results in Peutz-Jeghers Syndrome and is associated with neoplasias of the lung, pancreas, and breast. Based on preliminary analysis, overexpression of LKB1 by shRNA in TNBC cell lines results in suppression of EMT and reduction of the cancer stem cell population. Additional studies show that LKB1 overexpression has no effect on growth rate in 2D culture while significant reduction in 3D mammosphere formations can be seen. Downstream studies using commercially available SIK1 inhibitor HG-9-91-01 is able to induce a larger fraction of CSC from reduced LKB1 overexpression as well as from baseline levels. DISCUSSION/SIGNIFICANCE OF FINDINGS: Overall, our results suggest that LKB1 acts through SIK1 to suppress EMT and the generation of cancer stem cells. This results in reduced cancer functionality, as evidenced by inhibition of mammosphere formation. These results establishes a foundation for future mechanistic studies on the LKB1 axis and its mechanisms in TNBC.

Background: As carbapenem-resistant Enterobacteriaceae (CRE) prevalence increases in the United States, the risk of cocolonization with multiple CRE may also be increasing, with unknown clinical and epidemiological significance. In this study, we aimed to describe the epidemiologic and microbiologic characteristics of inpatients cocolonized with multiple CRE. Methods: We conducted a secondary analysis of a large, multicenter prospective cohort study evaluating risk factors for CRE transmission to healthcare personnel gown and gloves. Patients were identified between January 2016 and June 2019 from 4 states. Patients enrolled in the study had a clinical or surveillance culture positive for CRE within 7 days of enrollment. We collected and cultured samples from the following sites from each CRE-colonized patient: stool, perianal area, and skin. A modified carbapenem inactivation method (mCIM) was used to detect the presence or absence of carbapenemase(s). EDTA-modified CIM (eCIM) was used to differentiate between serine and metal-dependent carbapenemases. Results: Of the 313 CRE-colonized patients enrolled in the study, 28 (8.9%) were cocolonized with at least 2 different CRE. Additionally, 3 patients were cocolonized with >2 different CRE (1.0%). Of the 28 patients, 19 (67.6%) were enrolled with positive clinical cultures. Table 1 summarizes the demographic and clinical characteristics of these patients. The most frequently used antibiotic prior to positive culture was vancomycin (n = 33, 18.3%). Among the 62 isolates from 59 samples from 28 patients cocolonized patients, the most common CRE species were Klebsiella pneumoniae (n = 18, 29.0%), Escherichia coli (n = 10, 16.1%), and Enterobacter cloacae (n = 9, 14.5%). Of the 62 isolates, 38 (61.3%) were mCIM positive and 8 (12.9%) were eCIM positive. Of the 38 mCIM-positive isolates, 33 (86.8%) were KPC positive, 4 (10.5%) were NDM positive, and 1 (2.6%) was negative for both KPC and NDM. Also, 2 E. coli, 1 K. pneumoniae, and 1 E. cloacae were NDM-producing CRE. Conclusion: Cocolonization with multiple CRE occurs frequently in the acute-care setting. Characterizing patients with CRE cocolonization may be important to informing infection control practices and interventions to limit the spread of these organisms, but further study is needed.

Funding: None

Disclosures: None

We describe a hybrid pixel array detector (electron microscope pixel array detector, or EMPAD) adapted for use in electron microscope applications, especially as a universal detector for scanning transmission electron microscopy. The 128×128 pixel detector consists of a 500 µm thick silicon diode array bump-bonded pixel-by-pixel to an application-specific integrated circuit. The in-pixel circuitry provides a 1,000,000:1 dynamic range within a single frame, allowing the direct electron beam to be imaged while still maintaining single electron sensitivity. A 1.1 kHz framing rate enables rapid data collection and minimizes sample drift distortions while scanning. By capturing the entire unsaturated diffraction pattern in scanning mode, one can simultaneously capture bright field, dark field, and phase contrast information, as well as being able to analyze the full scattering distribution, allowing true center of mass imaging. The scattering is recorded on an absolute scale, so that information such as local sample thickness can be directly determined. This paper describes the detector architecture, data acquisition system, and preliminary results from experiments with 80–200 keV electron beams.

Obesity, insulin resistance and disturbed glucose metabolism cluster within the Insulin Resistance Syndrome (IRS). Whether this reflects shared genetic or environmental factors detectable in ‘normal’ populations (not selected for IRS features) is unknown. This study estimated (i) genetic influences on IRS traits and (ii) shared and specific genetic and environmental factors on the relationships between these traits in healthy female twins. Fasting insulin, glucose, total and central fat were measured in 59 monozygotic (MZ) and 51 dizygotic (DZ) female twin pairs aged ( ± SD) 52 ± 13 years. Body fat was measured by dual-energy X-ray absorptiometry, insulin resistance and secretion by a modified homeostasis model assessment. Using intraclass correlation coefficients and univariate model-fitting analyses, genetic influences were found in total fat, central fat, insulin resistance, fasting glucose and insulin secretion, with genetic factors explaining 64, 57, 59, 75 and 68% of their variance, respectively, using the latter technique. In matched analysis intra-pair differences in total and central fat related to intra-pair differences in insulin resistance (r2 = 0.19, P < 0.001). Multivariate model-fitting showed a close genetic relationship between total and central fat (r = 0.88). The genetic correlation between IR and central fat (0.41) was significantly greater than that for total fat (0.24), suggesting that central fat is not only a predictor of, but shares considerable genetic influence with, insulin resistance. In Cholesky analysis, these genetic influences were separate from those shared between central and total fat. In conclusion, both shared and specific genetic factors regulate components of the IRS in healthy females. However, there were discrete genetic influences on -cell insulin secretion, not shared with other IRS components, suggesting that a separate genetic propensity exists for Type2 diabetes. These findings suggest we may understand the genetic and environmental influences on IRS from the study of the normal population.

Echocardiography is critical in the assessment of patients with hypoplastic left heart syndrome. Fundamental techniques and standardised approaches are useful when evaluating patients with hypoplastic left heart syndrome prenatally, after birth, and before the Norwood operation (Stage 1); after the Norwood operation, before and after the superior cavopulmonary anastomosis (Stage 2); before and after the Fontan operation (Stage 3); and for chronic surveillance after the Fontan operation. From foetal assessment to ongoing surveillance after the Fontan procedure, echocardiography remains the primary technique for cardiac monitoring in this growing population of children and adults.