141P - An unconditionally dynamic culture system for cancer stem cell

Background

The cultivation mode for tumor stem cells generally involves sorting. Fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are the main techniques for sorting CSCs. However, this is not the optimal solution. FACS is time-consuming, technically demanding, and yields a limited number of sorted cells. MACS, while operationally simple, can only sort one marker at a time. Conditional culture media require the addition of a large quantity of expensive growth factors. We established a dynamic suspension culture without additional sorting and cytokines to obtain cancer stem cell.

Methods

A dynamic suspension culture system without sorting and additional cytokines was used to obtain cancer stem cells. The immunostaining, RNA-seq, metabolomics and proteomics were performed to analyze the properties of cancer stem cells.

Results

The cancer stem cell was successfully cultivated from unconditionally dynamic suspension culture system. They exhibited the expression of specific cancer stem markers, such as upregulated CD44, CD133, ALDHA1, ABCB5 and decreased CD24.These cells possessed self-renewal capability and strong tumorigenicity. Meanwhile, their transcriptome, metabolism, proteins were reprogrammed. Drug metabolism cytochrome P450 and resistance to many drugs, such as platinum and methotrexate were activated. A metabolic switch from glycolysis to oxidative phosphorylation was founded in these cells. Importantly, actin cytoskeleton rearrangement and epithelial-mesenchymal transition were observed in these cells. The RNA-seq analyses show that signaling pathways, including HIF, Hippo and Wnt pathway were activated, resulting in the remodeling of cancer stem cell.

Conclusions

Independently sorting and the addition of cytokines, a dynamic suspension culture system can successfully generate cancer stem cells. These cells exhibited typical cancer stem cell properties, including reprogrammed transcriptome, metabolism, and protein levels. This unconditional dynamic suspension culture system offers an alternative method for cultivating cancer stem cells and presents a novel model for exploring their characteristics.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.