Visualizing the cancer stem‐like properties of canine tumour cells with low proteasome activity

Abstract

Cancer stem-like cells (CSCs) cause treatment failure in various tumours; however, establishing CSC-targeted therapies has been hampered by difficulties in the identification and isolation of this small sub-population of cells. Recent studies have revealed that tumour cells with low proteasome activity display a CSC phenotype that can be utilized to image CSCs in canines. This study visualizes and reveals the CSC-like properties of tumour cells with low proteasome activity in HMPOS (osteosarcoma) and MegTCC (transitional cell carcinoma), which are canine cell lines. The parent cells were genetically engineered to express ZsGreen1, a fluorescent protein connected to the carboxyl-terminal degron of canine ornithine decarboxylase that accumulates with low proteasome activity (ZsG + cells). ZsG + cells were imaged and the mode of action of this system was confirmed using a proteasome inhibitor (MG-132), which increased the ZsGreen1 fluorescence intensity. The CSC-like properties of ZsG + cells were evaluated on the basis of cell divisions, cell cycle, the expression of CSC markers and tumourigenicity. ZsG + cells underwent asymmetric divisions and had a low percentage of G0/G1 phase cells; moreover, ZsG + cells expressed CSC markers such as CD133 and showed a large tumourigenic capability. In histopathological analysis, ZsG + cells were widely distributed in the tumour samples derived from ZsG + cells and in the proliferative regions of the tumours. The results of this study indicate that visualized canine tumour cells with low proteasome activity have a CSC-like phenotype and that this visualization system can be utilized to identify and isolate canine CSCs.

Regulation of programmed death ligand 1 expression by interferon‐γ and tumour necrosis factor‐α in canine tumour cell lines

“…The qPCR conditions were as follows: initial denaturation at 95 C for 2 min, followed by 40 cycles of denaturation at 95 C for 15 s, annealing at 60 C for 20 s, and extension at 72 C for 1 min. All relative mRNA expressions were normalized against the reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), [37][38][39][40][41] and quantified using the delta-delta Ct method. 42 The sequences of the primers used in the experiments [41][42][43][44][45][46][47][48] are listed in Table 1.…”