48PDevelopment of MDA-MB-231-3D-Spheroid as a reliable model for studying Nav1.5 and nNav1.5-mediated breast cancer metastasis

Background Nav1.5 and nNav1.5 are potent metastatic markers for breast cancer with aggressive phenotype; detected in tumour tissues positive for lymph node metastasis. In vitro, most of the studies involving Nav1.5 and nNav1.5 were evaluated in monolayer cultured cells. Subsequently, since 3D-spheroid culture is now known for its ability to mimic multiple in vivo tumour microenvironment, there is a need to re-evaluate the potential role of Nav1.5 and nNav1.5 in a reliable tumour-accurate model. This study aimed to develop MDA-MB-231-3D-spheroid culture and assess the mRNA gene expression of Nav1.5 and nNav1.5 including several other potent markers for aggressive breast cancer, MMP1, MMP13 and fibronectin. Methods MDA-MB-231-3D-spheroids were cultured using scaffold-free liquid overlay method (via agarose layered 96-well flat bottomed plate) for 15 days. The total RNA was conventionally extracted and converted to cDNA. The mRNA expression for Nav1.5 and nNav1.5, MMP1, MMP13 and fibronectin were measured using real-time PCR and the expression were analysed via the 2-ΔΔCt method in which the monolayer culture was used as a normalizing control. Results MDA-MB-231-3D-spheroid was successfully developed. mRNA expression of Nav1.5, nNav1.5, MMP1 and fibronectin were significantly higher (p Conclusions Evaluating the role of tumour markers in a reliable tumour-accurate model is very important. MDA-MB-231-3D-spheroid was able to increase the expression of Nav1.5 and nNav1.5 and can be utilized to re-evaluate the role of Nav1.5 and nNav1.5 in cancer metastasis. Legal entity responsible for the study Institute for Research in Molecular Medicine, Universiti Sains Malaysia. Funding Universiti Sains Malaysia, FRGS (203/CIPPM/6171212). Disclosure All authors have declared no conflicts of interest.