PO-307 Differential secretome analysis of cancer-associated adipocytes (CAA) and mature adipocytes to identify adipocyte-driven micro-environmental regulators of breast cancer progression

Abstract

IntroductionIn breast cancer, obesity is linked to invasive tumours that respond poorly to chemotherapy. We have explored relationships between breast cancer cells and adipocytes in the tumour microenvironment using a transwell co-culture system. Human breast adipocytes induced chemo-resistance in both hormone receptor positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells. In addition, cancer-associated adipocytes (CAA) induced a partial epithelial-mesenchymal transition, creating a population of breast cancer cells that were highly migratory. In this study, we used discovery mass spectrometry and antibody arrays to analyse the proteins secreted by CAA to identify proteins that may contribute to breast cancer progression.Material and methodsBreast adipose tissue samples were collected from patients undergoing breast surgery at Christchurch Hospital. Pre-adipocytes were isolated and differentiated into mature adipocytes, followed by co-culture with MCF-7 and MDA-MB-231 cancer cells for 3 days in a transwell co-culture system. Breast cancer cells were removed and adipocytes were washed and serum free media was added for 24 hours. This conditioned media was collected from pre-adipocytes, mature differentiated adipocytes and CAA co-cultured with either MCF-7 or MDA-MB-231 cells. Secretomes were compared using antibody arrays and iTRAQ labelling with LC-coupled LTQ-Orbitrap tandem mass spectrometry. Validation of iTRAQ results was performed using ELISA.Results and discussionsOf the 327 proteins identified using iTRAQ-MS, 11.3% were intracellular and thus excluded; the remainder were either extracellular or secreted proteins. Of the 183 secreted proteins, 45% of these were secreted at higher levels by adipocytes after co-culture with breast cancer cells compared with adipocytes alone. These included proteins associated with inflammation and matrix remodelling. Chitinase 3-like 1 (Chi3L1) levels were increased according to both iTRAQ-MS and antibody array analysis. Recombinant Chi3L1 treatment (4 ng/ml) increased viability, proliferation, migration and resistance to chemotherapy in both breast cancer cell lines.ConclusionOur study describes for the first time, the secretome profile from CAA and provides a comprehensive platform for further research investigating local interactions between breast cancer cells and CAA. Our data identified CAA derived Chi3L1 as a key player in the interactions between adipocytes and cancer cells within the tumour microenvironment.