Abstract 99P
Background
Cancer stem cells (CSCs) are characterized by their enhanced ability to initiate tumor growth, proliferate, invade, migrate, and resist therapeutic treatments. However, there are no specific markers or optimal methods available for culturing CSCs. We developed a dynamic suspension culture method that eliminates the need for sorting and cytokines, enabling the efficient isolation of CSCs.
Methods
A dynamic suspension culture system, without sorting or additional cytokines, was used to generate CSCs. The properties of these cells were analyzed through immunostaining, RNA sequencing, metabolomics, and proteomics. A mouse model was established to detect the immune microenvironment by single cell RNA sequencing.
Results
Cancer spheroids were successfully cultivated from unconditionally dynamic suspension culture system. They exhibited the expression of 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, RhoA-activated actin cytoskeleton rearrangement and epithelial-mesenchymal transition were observed in these cells. Hippo pathway was activated via reduced p-YAP and TAZ, 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. Cancer spheroids exhibited typical cancer stem cell properties, including reprogrammed transcriptome, metabolism, and protein levels. CD44 Reprograms Cellular Metabolism to drive tumorigenesis and cancer stemness in cancer spheroids via the RhoA-activated YAP-TAZ pathway. This unconditional dynamic suspension culture system offers an alternative method for cultivating cancer stem cells and presents a novel model for exploring their characteristics.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Has not received any funding.
Disclosure
All authors have declared no conflicts of interest.