Roccuzzo, M. | Search bioRxiv

bioRxiv2024

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The intrinsic biocompatibility, stability in biofluids, and heterogeneous molecular cargo of EVs promise advancements in targeted therapy applications. However, predicting cell-targeting spectrum and cargo delivery are fundamental challenges for exploiting EVs or hybrid formulations. In this work, we combined cell-based and biochemical approaches to understand if secreted EVs show predictable EV-cell interactions and consequent cargo delivery. We exploited the tetraspanin CD81 to encode full-length recombinant proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. These fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs when transiently over-expressed in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used in EV-cell interaction assays. In time-frames where the global GFP spot distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 manipulation in isogenic cells remarkably affected the tropism of heterologous EVs. In this line, secreted doxorubicin-EVs, which showed improved efficacy compared to the free drug, had a reduced cell-killing activity on SK-BR-3 with a knocked-out HER2 receptor. Interestingly, the fusion protein-corresponding transcripts also present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, as detected by ddPCR in tissue biopsies, improving our sensitivity in detecting bioavailable cargoes. These data show multiple mechanisms underlying EV-cell interactions and prioritize the profiling of surfaceomes for better comprehension of cell engagement and design new generations of EV-based nanovehicles.

molecular biology↗

bioRxiv2018

Precursor miRNAs are trafficked along axons associated with vesicles and locally processed to regulate growth cone steering

Various species of non-coding RNAs (ncRNAs) are enriched in subcellular compartments but the mechanisms orchestrating their delocalization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked, anchored to CD63-positive vesicles, to distal axons along microtubules. Upon exposure to the chemotropic cue Sema3A, pre-miRNAs are processed specifically within axons into newly synthesized mature miRNAs, which, in turn, silence the basal translation of TUBB3 but not of APP. At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly synthesized miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodelling of subcellular compartments.\n\nHighlightsO_LIPrecursor miRNAs are actively transported along axons to the growth cone tethered to CD63-positive vesicles\nC_LIO_LISema3A but not Slit2 induces the local biogenesis of specific miRNAs within axons\nC_LIO_LIMature miRNAs are important for growth cone responsiveness ex vivo and the establishment of functional connections in vivo\nC_LIO_LINewly synthesized miRNAs inhibit the basal translation of TUBB3 but not APP upon Sema3A exposure\nC_LI

cell biology↗