Evaluation of mouse polyclonal antibody against nNav1.5 to recognize nNav1.5 protein in human breast cancer cells using fluorescence immunostaining

Abstract

Background: Neonatal splice variant of Nav1.5 (nNav1.5) is a known tumor-associated antigen (TAA), re-expressed in adult aggressive breast cancer/tumor biopsies positive for lymph node metastasis. Accurate assessment of nNav1.5 protein in breast cancer cells/tissue is important in validating its potential value as a prognostic or predictive marker. Mouse polyclonal antibody (pAb) against nNav1.5 has been obtained. Herein, the ability of the pAb to selectively recognize nNav1.5 protein was evaluated on a panel of cell lines using fluorescence immunostaining. Method(s): Mouse mammary cell line, 4T1, human breast cancer cell lines, MDA-MB-231, and MCF-7 as well as human non-cancerous epithelial cell line, MCF-10A were acquired (ATCC) and maintained. Accordingly, the cells were cultured in its respective complete media supplemented with 5% fetal bovine serum (FBS), L-glutamine (4mM) and penicillin-streptomycin (1%) at 37 degreeC in a 5% CO 2, relatively humidified atmosphere. For fluorescence immunostaining, the cells were seeded overnight on a cover-slip in 24-well culture plate, fixed, permeabilized and stained with primary pAb followed by incubation with secondary antibody conjugated with AlexaFluor-555. Fluorescence microscopy was conducted using Leica Inverted DMi8 Fluorescence and captured using DFC 365 FX Camera. Result(s): Red fluorescence (secondary AlexaFlour-555) signals were prominent in aggressive human breast cancer cells, MDA-MB-231 and aggressive mouse mammary cancer cells, 4T1; both cell lines are known as positive for nNav1.5 expression. On the contrary, in less aggressive human breast cancer cells, MCF-7 and non-cancerous human breast epithelial cells, MCF-10A which lacks nNav1.5 expression, red fluorescence signals were not detected similar to the no-primary and no-primary/secondary controls. Conclusion(s): The obtained mouse pAb against nNav1.5 could serve as a useful tool in validating nNav1.5 protein expression in breast cancer cells. Legal entity responsible for the study: The authors. Funding(s): Ministry of Energy, Science, Technology, Environment and Climate Change, Malaysia (Science Fund CIPPM/6113610). Disclosure: All authors have declared no conflicts of interest. Copyright © 2019 European Society for Medical Oncology