Biology subjects

Adem, B.

Publications and source records attributed to Adem, B..

3 recordsLinked to original sources

bioRxiv2026

TSPAN3 defines a distinct intracellular trafficking route to secretory multivesicular bodies

Extracellular vesicles (EVs) comprise molecularly diverse populations generated through multiple membrane-trafficking pathways, yet the intracellular basis of this heterogeneity remains poorly understood. Here, we identify the EV-associated tetraspanin TSPAN3 as a marker of a secretory multivesicular body (MVB) population that is molecularly and functionally distinct from canonical CD63-positive compartments. Using endogenous genome editing, live-cell and super-resolution microscopy, electron microscopy, quantitative EV secretion assays, and complementary proteomic approaches, we show that TSPAN3 localizes to fusion-competent MVBs but exhibits limited overlap with CD63 during secretion. Unlike CD63, which extensively traffics through the plasma membrane and depends on YXX{Phi}-mediated endocytic retrieval, TSPAN3 reaches secretory MVBs predominantly through an intracellular trafficking route that relies on a dileucine-containing sorting region. Orthogonal proximity-labeling and affinity-purification proteomics revealed that TSPAN3-positive compartments are associated with a selective LC3/ATG8-related membrane network, including GABARAPL2 and proteins involved in endosomal membrane remodeling and fusion. Perturbation of residues required for this association impaired localization to LC3-positive compartments and reduced secretory MVB fusion. Consistent with these findings, pharmacological disruption of autophagy- and endolysosomal-associated pathways differentially altered TSPAN3-positive EV secretion. Finally, proximity-labeled EV proteomics demonstrated that TSPAN3-associated EVs possess cargo signatures distinct from CD63-associated EVs, with greater representation of endosomal and endolysosomal proteins suggesting that tetraspanin-associated membrane nanodomains retain molecular signatures consistent with their intracellular trafficking history. Together, our findings identify TSPAN3 as a marker of a previously unrecognized secretory MVB population distinguished by its intracellular trafficking, molecular interactions, and EV composition, supporting a model in which distinct tetraspanin-organized membrane nanodomains are associated with different intracellular trafficking routes and molecularly distinct EV populations.

cell biology↗

bioRxiv2022

Resistance to EGFR inhibitors in lung cancer occurs through horizontal transfer and is associated with increased caveolins expression

Resistance to treatment is a major clinical problem and a major cause of cancer-related deaths. Understanding the biological basis of resistance acquisition is of utmost importance to improve the clinical management of cancer patients. NGS analysis of human lung cancer (LC) tumors from patients that relapsed after treatment with EGFR-tyrosine kinase inhibitors (TKI), revealed that the p.T790M resistance mutation is not present in all the relapsing tumor cells, suggesting that LC cells can become resistant even if not carrying the p.T790M mutation. Using in vitro treatments with conditioned medium (CM) and in vivo co-inoculation experiments, we show that LC cells sensitive to EGFR-TKIs (S cells) acquire resistance faster when treated with CM from LC cells resistant to EGFR-TKIs (R cells) or when co-inoculated with R cells in opposite flanks of the same animal. Importantly, we show that acquisition of resistance is not due to the emergence of subpopulations of cancer cells with new resistance mutations. Using transcriptomics, we show that acquisition of resistance is associated with upregulation of genes involved in endocytosis, namely caveolins CAV1 and CAV2. These findings were validated in human clinical samples, where an increase in CAV1 and CAV2 expression was associated with tumor relapse after treatment with EGFR-TKIs. Our results suggest that acquisition of resistance to targeted therapies results from the combined effect of selection of cells harboring specific resistance mutations and horizontal transfer of the resistance phenotype. These findings may pave the way to bring intercellular communication into the realm of cancer treatment. One Sentence SummaryResistance to EGFR inhibitors is transferred horizontally between lung cancer cells and is associated with gain of expression of caveolins.

cancer biology↗

bioRxiv2021

ExoBow: A transgenic strategy to study CD63 exosomes in vivo

Exosomes are described as central players in a myriad of biological processes. However, the available methodologies to study their function in complex biological systems in vivo are still very limited. The biodistribution of endogenously produced exosomes, the ability to trace their spontaneous flow in order to identify the cell types they interact with, remains a major challenge. New tools to identify comprehensive networks of communication established by exosomes originated in distinct cell types in vivo, are fundamental for a better understanding of their biology. Here, we describe the development of a genetically engineered mouse model that allows the expression of the mouse CD63 exosomal marker fused with one (monocolor) or up to four fluorescent proteins (multireporter), the ExoBow. The genetic design of the ExoBow transgene allows the conditional expression of the reporters in any tissue/cell-type in an inducible or non-inducible fashion. In addition, communication mediated by CD63 positive (CD63+) exosomes can be identified amongst the same tissue/cell types using the multireporter version of the model, in order to map intra-organ/tissue communication. We demonstrate the applicability of the ExoBow transgene in normal physiological conditions and in the context of cancer, using pancreas as a working model. The ExoBow comprises a unique strategy to identify intra- and inter-organ/cell-type communication mediated by CD63+ exosomes. We believe this tool will contribute for a better understanding of the complex interactions occurring in vivo that underly the biology of exosomes in health and disease.

cell biology↗