Intracellular mRNA phase separation induced by cationic polymers for tumor immunotherapy

Abstract

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. Graphical Abstract

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding

“…Because of their accessibility and stability, chemically synthesized or PCR-made probes are currently preferred to in vitro -transcribed ones. In the most modern application within the field of RNA condensation, FISH has been deployed, among other uses, to detect the phenomenon of RNA microsatellite repeat-driven LLPS to regulate oncogene transcription, to track the chemical modulation of RNA LLPS, and to localize a target RNA within the outer shell of ribonucleoprotein coacervates . Quantitative information can also be extracted by analyzing fluorescence intensity in condensed RNA regions, while combining FISH with time-lapse imaging can enable the observation of dynamic changes in RNA granules over time.…”

The Burgeoning Significance of Liquid-Liquid Phase Separation in the Pathogenesis and Therapeutics of Cancers

“…Nudix hydrolase 5 (NUDT5), crucial for ATP synthesis in breast cancer cell nuclei, is targeted by TH5427, a specific inhibitor that disrupts nuclear ATP generation, impacting hormone-induced chromatin remodeling and progestin-dependent gene regulation 121 . Studies using a mouse breast cancer model have highlighted that cationic polymers, like cDex and DETA-Dex, can impede RNA LLPS, thereby inhibiting TGFβ1 mRNA translation in tumor cells 122 . Moreover, Hu et al identified that the cell-polarity protein Par3 facilitates the phase separation of junctional adhesion molecule-A during breast cancer metastasis, altering osmotic pressure and mechanical properties 123 .…”

Rational design of polymer-based mRNA delivery systems for cancer treatment

“…[137][138][139][140][141] RNA or RNArelated liquid-liquid phase separation methods may be used to provide novel approaches to regulating TME. 142 Xing et al 143 introduced cationic polymers into living cells, which were then combined with negatively charged TGF-β1 mRNA, and liquid-liquid phase separation occurred, preventing the translation of TGF-β1 mRNA. As a result, the immunosuppressive ability of tumor cells in TME was reduced, triggering significant anti-tumor responses and improving the efficiency of tumor immunotherapy.…”