Nanoparticle's Encapsulation Ability Is More Efficient for Characterization of Invading Breast Cancer Cells Than EMT Markers

PURPOSE Metastasis is leading cause of cancer death. The essential step of metastasis is invasion. The actin cytoskeleton plays a pivotal role in invasion and is closely linked to endocytosis process. We previously distinguished highly and lowly invasive breast cancer (BC) cell lines based on their tendency to encapsulate 100-200 nm nanoparticles. Epithelial-to-mesenchymal transition (EMT) primarily initiates invasion, with EMT marker expression often used to gauge tumor invasiveness. The Boyden chamber migration assay serves as the gold standard for assessing tumor invasiveness, measuring cancer cells' migration towards a chemoattractant through subcellular pores. This study aims to characterize BC cell subpopulations, separated by the Boyden chamber, based on nanoparticle encapsulation and EMT marker expression. METHODS The highly invasive (HI) and lowly invasive (LI) BC cell lines (MDA-MB-231 and MCF-7, respectively) were separated into migrated and non-migrated subpopulations by Boyden chambers with 8µm membrane pores for 72 h. To assess encapsulation ability, cells were incubated with 200nm fluorescent carboxylated nanoparticles and nuclei stain for 1 hour, extensively washed with PBS and trypsin, and fixed. The images were captured by fluorescent microscopy, and nanoparticle colocalization with cells was quantified using Pearson`s coefficients. The standard antibody staining protocols were utilized for EMT markers: E-cadherin, N-cadherin, Vimentin and Snail1/2. The fluorescence intensity per image for each marker was normalized, and statistical significance was determined by ANOVA analysis. RESULTS The subpopulations of migrated cells were 32% and 2.2% for HI and LI cell lines, respectively. The significant differences between the expression of EMT markers in subpopulations were observed only in vimentin of HI cells (5% difference) and E-cadherin of LI (11% difference). At the same time, the differences in encapsulation efficiency between separated subpopulations of HI and LI cells were 30 % and 43%, respectively. CONCLUSION The EMT markers were found to be able to differentiate HI and LI cell lines, however they were not effective for distinguishing subpopulations within the same cell line. In contrast, the subpopulations can be clearly differentiated by the efficiency of nanoparticles encapsulation.