Enrichment of cancer stem-like cells by the induction of epithelial-mesenchymal transition using lentiviral vector carrying E-cadherin shRNA in HT29 cell line

Abstract

A better understanding of cancer stem cells (CSCs) may facilitate the prevention and treatment of cancers. Epithelial-mesenchymal transition (EMT) is a process activated during invasion and metastasis of tumors. EMT induction in normal and tumor cells makes them more resistant to chemotherapy. E-cadherin is a membrane protein and plays a role in tumor invasion, metastasis, and prognosis. Downregulation of E-cadherin is a hallmark of EMT. Here, we created a model of cancer stem-like cells enrichment via EMT induction using E-cadherin downregulation in HT29 cell line using a lentiviral vector carrying shRNA. We aimed to evaluate cancer and anti-CSC chemotherapeutics screening. The markers of EMT and CSCs were assessed and compared with control cells using flow cytometry, real-time PCR, immunocytochemistry, western blot, migration assay, invasion assay, and colony formation assay. The transduced cells showed a mesenchymal morphology. High levels of EMT-related proteins were also expressed. These results confirmed that the transduced cells underwent EMT. In addition, we observed an increased population of E-cadherin-downregulated HT29 cell line among the cells expressing colon CSC markers (CD133⁺ and CD44⁺ ) after EMT induction. E-cadherin-downregulated cells were morphologically like mesenchymal cells, and the number of CD133⁺ - and CD44⁺ -cells (CSC-like cells) increased. These cells can be used as stable models to study cancer cells and screening of antitumor therapeutics.

Keywords: E-cadherin; cancer stem cells (CSCs); cancer therapy.; epithelial-mesenchymal transition (EMT); lentiviral vector.