Intracellular mRNA phase separation induced by cationic polymers for tumor immunotherapy

The formation of biomolecular condensates via liquid‒liquid phase separation (LLPS) is an advantageous strategy for cells to organize their subcellular compartments for diverse functions. Recent findings suggest that RNA or RNA-related LLPS techniques have potential for the development of new cellular regulation strategies. However, manipulating RNA LLPS in living cells has great challenges. Herein, we report that cationic polymers (CPs) have strong RNA LLPS-inducing activity. By introducing CPs into living cells or RNA solutions, significant RNA LLPS was verified through confocal imaging, turbidity assays, and fluorescence recovery after photobleaching (FRAP) tests. Among them, turbidity kinetics determinations indicated that the hydrophilic positively charged amino groups on the CPs play essential roles in RNA phase separation. Moreover, the LLPS induced by the cationic polymers dramatically changed the gene expression patterns in the cells. Interestingly, we found that TGFβ1 mRNA was highly encapsulated in the RNA droplets, which lowered the immunosuppressive capability of the tumor cells and triggered marked antitumor reactions in a mouse breast cancer model. Thus, we present here the CP-based modulation of RNA LLPS as a novel transcriptional manipulation method with potential for cancer immunotherapy drug development.