Introduction

Existence of cancer stem-like cells (CSCs) has long been proposed as a cause of treatment failure in many types of tumors. However, studies to target CSCs have been hampered by difficulty in identifying this small subpopulation of cells. Recent research to characterize CSCs have revealed that CSCs have low proteasome activity, which could be utilized for CSCs visualization. The purpose of this study was to visualize CSCs in a canine osteosarcoma cell line.

Methods

A canine osteosarcoma cell line (HMPOS) was used for this study. ZsGreen1 and carboxyl-terminal region of ornithine decarboxylase genes were transfected into the parent cells using a lentiviral system, accumulating the fluorescence in the cells with low proteasome activity, ZsG+ cells. The CSC-like properties of ZsG+ cells were evaluated on self-renewal capacity, function of ABCG2 transporter, differentiation capacity, cell cycle, and expression level of CSC markers. After irradiation, radio-sensitivity was examined, evaluating clonogenic assay, and p53-binding protein 1 (53BP1) foci formation.

Results

The accomplishment of the transfection was confirmed with inhibition of proteasome activity, the fluorescent intensity of most cells increased in HMPOS-ZsG. The CSC-like properties of ZsG+ cells showed higher self-renewal capacity, function of ABCG2 transporter, differentiation capacity, and expression level of CSC markers compared with non-ZsG+ cells. The cell cycle analysis showed low G0/G1 and high S phase of ZsG+ cells. After irradiation, ZsG+ cells were resistant against X-ray irradiation.

Conclusion

CSCs in HMPOS cells could be successfully visualized by ZsGreen1 system.