Abstract P5-02-05: Optimizing the differentiation of the 3T3-L1 MBX mouse cell line to mature and obese adipocytes for in vitro breast cancer studies

Abstract

Abstract White adipocytes are considered an endocrine organ. Present in breast tissue, they crosstalk in a paracrine manner with breast cells, secreting signals into the tumor microenvironment to drive cancer development as well as metastasis. 3T3-L1, a mouse fibroblast cell line, is widely used as an in vitro experimental model of white adipocytes for fat cell and cancer cell interaction studies. 3T3-L1 can be differentiated from the fibroblast cell line to adipocytes using a pharmacological cocktail of dexamethasone, methyl-3-isobutyl xanthine (IBMX) and insulin. Recently, ATCC has derived a clone from 3T3-L1 (ATCC CL-173), 3T3-L1 MBX, which has been claimed to have close to 100% differentiation. This 3T3-L1 MBX cell line has been recommended as a more suitable model for obesity research. However, there are few reported results describing their optimized differentiation conditions and/or any fat-cancer cell interactions. The current study has explored and optimized the concentrations of the differentiation cocktail for 3T3-L1 cells (ATCC CL-173) to differentiate its cloned derivative 3T3-L1 MBX. In the pharmacological cocktail, rosiglitazone was added as the 4th component as a prodifferentiative agent. Furthermore, the study has documented the differentiation of 3T3-L-MBX in the presence of the above-mentioned pharmacological cocktail with fatty acid combinations (palmitic acid, oleic acid, linoleic acid), to determine its applicability as an in vitro experimental model of obese adipocytes. The optimized concentrations of fatty acids that yielded the highest cell viability of 3T3-L1 MBX was determined using MTT assay. Additionally, the optimized combination of fatty acids was confirmed using oil-red-o adipogenesis staining. The adipocytokine levels of all the above mentioned in-vitro adipogenesis phases was evaluated using a mouse adipokine array kit as well as comparing the level of adipocytokines with those of fat tissue harvested from 6 month and 12 months old obese C3Hhej mice. Lastly, the study evaluated viability of three types of breast cells (non-tumorigenic, low metastatic, and highly metastatic) in the presence of conditioned medium collected from in-vitro 3T3-L1 MBX differentiated mature and obese adipocytes using an Annexin V-FITC assay kit. The study has found 3 days differentiation cycle of 3T3-L1 MBX with the addition of specific concentration of IBMX (0.5 mM), dexamethasone (1 µM), insulin (10 µg/ml) and rosiglitazone (2 µM) to be promising for its maximum differentiation. Further, 3T3-L1 MBX differentiated in presence of fatty acid were characterized to have higher lipid accumulation and adipokine secretion than the normally differentiated adipocytes. Citation Format: Noshin Mubtasim, Dr. Lauren Gollahon. Optimizing the differentiation of the 3T3-L1 MBX mouse cell line to mature and obese adipocytes for in vitro breast cancer studies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-05.