Elasticity Measurement of High-Metastatic Breast Cancer Cells That Highly Express Intermediate Filament Protein Nestin

Abstract

Nestin is an intermediate filament protein that is used for a neural stem cell marker. Since its high expression was reported in several high-metastatic cancer cells, nestin is considered to be involved in metastasis of cancer cells1. However, relationship between nestin and metastatic mechanism of cancer cells is unknown. Cancer cells are generally softer than benign cells2. Because intermediate filaments contribute to cell mechanical property by binding with other cytoskeletal proteins, nestin expression could affect elastic modulus of cancer cell. Previously, we have obtained a highly metastatic murine breast cancer cell line, SC2 which expressed three-fold nestin of parent strain. In this study, we established nestin-knockout SC2 cell by CRISPR/Cas9-mediated genome editing to evaluate an effect of nestin disruption on the cell. Intermediate filaments are composed by three domains and rod domain in the N-terminus is necessary to form bundles. In order to disrupt nestin gene, sgRNA was designed to target the rod domain of mouse nestin. Plasmid expressing the sgRNA and Cas9 was transfected into the SC2 cells by lipofection. After transfection, the cells were cultured in RPMI 1640 medium including G418 (500 μg/mL) for 10 days. Obtained clones were confirmed by immunostaining and three clones were found to be disrupted nestin. Analysis of genome sequence revealed that frameshift mutation was occurred by deletion of nucleotides in nestin gene of the obtained clones. These results indicated that nestin knockout succeeded by CRISPR/Cas9-mediated genome editing. To examine in vivo metastatic activity of nestin knockout cells, the SC2 and knockout cells were intravenously injected into mice. Survival time of mice injected with nestin-knockout cell was significantly prolonged, suggesting that nestin knockout reduced metastatic ability of the SC2 cells. We measured elastic modulus of cell body by using of atomic force microscopy (AFM) and cylindrical-shaped AFM probe with 2.2 μm in diameter. Force curve obtained when the probe was contacted with cell surface was fitted by Herz model and young’s modulus was calculated. Since the young’s modulus of the nestin-knockout cell was significantly higher than that of the SC2, nestin knockout caused increase in mechanical property. In the metastasis process, cancer cells invade surrounding tissue. Thus, expression of nestin reduces the elastic modulus in the cell body and leads high metastatic activity. (1) Kleeberger, W. et al. Cancer research 67, 9199-9206, 2007. (2) Cross, E. S. et sl. Nature nanotechnology 2, 780-783, 2007.