Isolation of cancer stem cells by verapamil selection from 4T1 mouse mammary cancer cell line

Abstract

In breast cancer, the tumor relapse and metastasis are the reasons for the ineffectiveness treatment of traditional therapy. Growing evidences suggest that cancer stem cells (CSCs) are responsible for this tumor progression. CSCs are presumed to have unlimited proliferative and self-renewal ability and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette (ABC) transporters. This research aims to enrich CSCs based on this increased expression of ABC transporters in anti-cancer drug assay. We figured out the verapamil concentration which specified for enrichment drug-resistant cells from 41T mouse mammary cancer cell line and investigated whether treated cells express functional characteristics of CSCs. In order to find out this suitable drug concentration, 4T1 cells were incubated in various concentrations of verapamil (10-100µg/ml). Then, real time PCRwould be performed with verapamil selected cellsto confirmoverexpression of multidrug-resistant gene (MDR) of these cells. After that, mammosphere generation and in vivo tumor formation in immunodeficient mice were also conducted to see if these cells carried the characteristics of CSCs.In the result, after selection in 50 µg/ml of verapamil, selected cells with the overexpression of MDR genehave increased properties of CSCs that are high invasion, mammospheres forming, and tumorigenic in mice. Keywords: cancer stem cell, verapamil, 4T1, resistance, breast cancer.