Evaluation for Inhibitory Effects of Hybrid Liposomes on the Growth of Breast Cancer Stem Cells using Soft Agar Colony Formation Assay

Abstract

We evaluated the inhibitory effects of nano-sized liposomal particles, hybrid liposomes (HLs), on the growth of breast cancer stem cells (BCSCs), and the effectiveness of soft agar colony formation assay for discriminating BCSCs. The flow cytometric (FCM) analysis of mouse-derived breast cancer (4T1-Luc) cells, using CD44v marker, inferred they contain 70% – 90% of BCSCs. In 4T1-Luc cells, the proportion of BCSCs remarkably decreased after adding HLs and was almost constant after adding doxorubicin (DOX). The 50% inhibitory concentration (IC₅₀) of DOX was 0.121 μM for 4T1-Luc cells. The 4T1-Luc cell number decreased after adding 5 μM DOX, without reducing BCSCs. After adding HL at concentrations above the IC₅₀ (99.6 μM) for 4T1-Luc cells, BCSCs gradually decreased to 37% of their concentration at 300 μM.

Furthermore, a reduction in BCSC proportion in HL-treated 4T1-Luc cells was observed using the soft agar colony formation method. Based on the HL-treated concentration, the live cell number significantly decreased and cell-survival was maintained with DOX treatment. Fusion and accumulation of the fluorescent phospholipid (NBDPC)-labeled HLs in these cells were analyzed using FCM and confocal laser scanning microscopy.

These results indicate that HL is more effective in suppressing the growth of BCSCs than DOX. The soft agar colony formation assay, which measures the tumorigenicity of cells in vitro, may be a promising discrimination method for the BCSCs, further reducing animal testing.