Publications

Despite the natural capacity of extracellular vesicles (EVs) to encapsulate intracellular compounds and transfer these to nearby or distant recipient cells, the intentional loading of EVs with cargo molecules remains a challenging endeavor. Pre-formation EV loading (i.e., during EV biogenesis), offers advantages compared to post-formation loading (i.e., after EV isolation), as EV integrity and composition are minimally perturbed. Pre-formation EV loading is primarily achieved through the genetic engineering of the producer cell, which is time consuming and not very flexible regarding the types of molecules that can be incorporated into EVs. In this work, we investigated the possibility of loading cargo molecules into EVs by delivering the cargo directly into the cytosol of the producer cells, which can subsequently be encapsulated into EVs as they are formed. For the cytosolic delivery of cargo molecules, we evaluated the use of photoporation. This membrane disruption technology has been demonstrated to successfully deliver a broad range of cargo molecules into virtually any cell type, while minimally impacting the cell's normal functioning and homeostasis. As a proof-of-concept, we delivered fluorescently labeled dextran macromolecules and anti-EGFP nanobodies into HEK293T cells genetically engineered with gag-EGFP fusion proteins, which are shuttled into EVs. Colocalization of cargo and EGFP fluorescence in secreted EVs can then serve as a convenient readout for successful EV loading. We established that photoporation had minimal impact on EV characteristics such as concentration, size, zeta potential and the enrichment of EV tetraspanin membrane surface molecules. We found that using EGFP-targeted nanobodies resulted in up to 53% loaded EVs (relative to the amount of EGFP EVs), while non-targeted dextran molecules produced on average 12% loaded EVs (relative to the amount of EGFP EVs). These results highlight the promise of photoporation for pre-formation loading of EVs.

JTD Keywords: Biogenesis, Challenge, Drug-delivery, Exosomes, In-vitro, Macromolecules, Microrna, Nanobubbles, Small interfering rna, Transferrin receptor

The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different geometries. For instance, the formation of multivesicular bodies (MVBs) and the biogenesis of extracellular vesicles both require the participation of the ESCRT-III sub-complex, and they differ in their initial membrane geometry before budding starts: the protein complex acts either from outside the membrane organelle (causing inward budding) or from within (causing outward budding). Several studies have reconstituted the action of the ESCRT-III subunits in supported bilayers and cell-sized vesicles mimicking the geometry occurring during MVBs formation (in-bud), but extracellular vesicle budding (out-bud) mechanisms remain less explored, because of the outstanding difficulties encountered in encapsulation of functional ESCRT-III in vesicles. Here, we provide a different approach that allows the recreation of the out-bud formation, by combining giant unilamellar vesicles as a membrane model and a microinjection system. The vesicles are immobilized prior to injection via weak adhesion to the chamber coverslip, which also ensures preserving the membrane excess area required for budding. After protein injection, vesicles exhibit outward budding. The approach presented in this work can be used in the future to disentangle the mechanisms underlying ESCRT-III-mediated fission, recreating the geometry of extracellular bud production, which remains a challenge. Moreover, the microinjection methodology can be also adapted to interrogate the action of other cytosolic components on the encapsulating membranous organelle. Copyright: © 2022 The Authors.

JTD Keywords: adhesion, budding, electroformation, escrt-iii, exosomes, extracellular vesicles, giant unilamellar vesicle (guv), light, microinjection, microparticles, plasma, Adhesion, Budding, Escrt-iii, Extracellular vesicles, Giant unilamellar vesicle (guv), Membrane, Microinjection

Neuroblastoma (NB) is the most common extracranial pediatric solid cancer originating from undifferentiated neural crest cells. NB cells express EZH2 and GLI1 genes that are known to maintain the undifferentiated phenotype of cancer stem cells (CSC) in NB. Recent studies suggest that tumor-derived extracellular vesicles (EVs) can regulate the transformation of surrounding cells into CSC by transferring tumor-specific molecules they contain. However, the horizontal transfer of EVs molecules in NB remains largely unknown. We report the analysis of NB-derived EVs in bioengineered models of NB that are based on a collagen 1/hyaluronic acid scaffold designed to mimic the native tumor niche. Using these models, we observed an enrichment of GLI1 and EZH2 mRNAs in NB-derived EVs. As a consequence of the uptake of NB-derived EVs, the host cells increased the expression levels of GLI1 and EZH2. These results suggest the alteration of the expression profile of stromal cells through an EV-based mechanism, and point the GLI1 and EZH2 mRNAs in the EV cargo as diagnostic biomarkers in NB.

JTD Keywords: exosomes, genes, lines, maintenance, pathway, proliferation, rna, stemness, tumor, Cancer

Cancer represents one of the conditions with the most causes of death worldwide. Common methods for its diagnosis are based on tissue biopsies—the extraction of tissue from the primary tumor, which is used for its histological analysis. However, this technique represents a risk for the patient, along with being expensive and time-consuming and so it cannot be frequently used to follow the progress of the disease. Liquid biopsy is a new cancer diagnostic alternative, which allows the analysis of the molecular information of the solid tumors via a body fluid draw. This fluid-based diagnostic method displays relevant advantages, including its minimal invasiveness, lower risk, use as often as required, it can be analyzed with the use of microfluidic-based platforms with low consumption of reagent, and it does not require specialized personnel and expensive equipment for the diagnosis. In recent years, the integration of sensors in microfluidics lab-on-a-chip devices was performed for liquid biopsies applications, granting significant advantages in the separation and detection of circulating tumor nucleic acids (ctNAs), circulating tumor cells (CTCs) and exosomes. The improvements in isolation and detection technologies offer increasingly sensitive and selective equipment’s, and the integration in microfluidic devices provides a better characterization and analysis of these biomarkers. These fully integrated systems will facilitate the generation of fully automatized platforms at low-cost for compact cancer diagnosis systems at an early stage and for the prediction and prognosis of cancer treatment through the biomarkers for personalized tumor analysis.

JTD Keywords: Cancer, Circulant tumor cells (CTC), Circulant tumor DNA (ctDNA), Exosomes, Liquid biopsy, Microfluidic, Sensors