Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models

The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation.

Establishment and analysis of conditional Rab1 and Rab5 knockout cells by using the auxin-inducible degron system

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. Since they have recently been shown to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, it has been extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed conditional KO (CKO) cells for Rab1 (Rab1A/1B) and Rab5 (Rab5A/5B/5C) by using the auxin-inducible protein degradation system. Rab1-CKO and Rab5-CKO led to eventual cell death >18 h and >48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and ER-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

EPCT-25. SMO PROTEIN DEPLETION IN SHH MEDULLOBLASTOMAS USING MICROBUBBLE-ENHANCED ULTRASOUND AND SIRNA LOADED CATIONIC NANOPARTICLES

RNA-based therapies offer unique advantages for treating pediatric brain tumors. However, the systemic delivery remains a major problem due to degradation of unmodified RNA in biological fluids, poor brain accumulation, and poor cancer cell uptake or escape from the endosomal lipid bilayer barrier. While nanoparticle encapsulation can prolong circulation time and facilitate cellular uptake, their accumulation in brain tumor remains particularly poor due to their low permeability across the blood-brain barrier and limited intratumoral penetration. Focused ultrasound, when combined with circulating microbubbles (MB-FUS) provides a physical method to transiently modulate the brain tumor microenvironment (TME) and improve nanoparticle delivery. Here, we have examined the delivery of siRNA targeting the Smoothened (SMO) pathway, packaged in 50 nm cationic lipid-polymer hybrid nanoparticles (cLPH:siRNA-SMO), combined with MB-FUS in murine SmoA2 sonic hedgehog (SHH) medulloblastoma. At 30 hours after treatment, we observed the depletion of the SMO protein target, responsible for driving SHH medulloblastoma formation and growth, in mice that had received treatment with MB-FUS and cLPH:siRNA-SMO, but not with cLPH:siRNA-SMO alone. We also confirmed that SMO protein depletion was spatially achieved in the tumor regions with detected cLPH:siRNA-SMO using FISH assay, and that there was 15 fold induction of tumor cell apoptosis compared to tumors in mice that had received cLPH:siRNA-SMO alone. The limited induction of apoptosis was observed with either cLPH:siRNA (non-targeting) or MB-FUS and cLPH:siRNA (non-targeting), suggest that the observed apoptosis induction in the SmoA2 model was the direct result of SMO depletion rather than nonspecific effects of MB-FUS or cLPH:siRNA. Our findings provide a paradigm shift in drug delivery in brain tumors, where physical methods and nanotechnology are tuned together to develop rational strategies for the effective delivery of nucleic acids in brain tumors.

A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

The OsTIR1/auxin-inducible degron (AID) system allows “on demand” selective and reversible protein degradation upon exposure to the phytohormone auxin. In the current format, this technology does not allow to study the effect of acute protein depletion selectively in one phase of the cell cycle, as auxin similarly affects all the treated cells irrespectively of their proliferation status. Therefore, the AID system requires coupling with cell synchronization techniques, which can alter the basal biological status of the studied cell population. Here, we introduce a new AID system to Regulate OsTIR1 Levels based on the Cell Cycle Status (ROLECCS system), which induces proteolysis of both exogenously transfected and endogenous gene-edited targets in specific phases of the cell cycle. This new tool paves the way to studying the differential roles that target proteins may have in specific phases of the cell cycle.

A polarity pathway for exocyst-dependent intracellular tube extension

Lumen extension in intracellular tubes can occur when vesicles fuse with an invading apical membrane. Within theCaenorhabditis elegansexcretory cell, which forms an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane and is required for its outgrowth, suggesting that exocyst-targeted vesicles extend the lumen. Here, we identify a pathway that promotes intracellular tube extension by enriching the exocyst at the lumenal membrane. We show that PAR-6 and PKC-3/aPKC concentrate at the lumenal membrane and promote lumen extension. Using acute protein depletion, we find that PAR-6 is required for exocyst membrane recruitment, whereas PAR-3, which can recruit the exocyst in mammals, appears dispensable for exocyst localization and lumen extension. Finally, we show that CDC-42 and RhoGEF EXC-5/FGD regulate lumen extension by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a pathway that connects CDC-42, PAR proteins, and the exocyst to extend intracellular tubes.