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Third Featured Abstract at ISEV 2020 Virtual Annual Meeting Reports That Expression of Tetraspanin 8 (Tspan8) Causes Nuclear Proteins to Be Incorporated into Tumor-Derived Extracellular Vesicles (EVs); Results Suggest New Mechanism of Action for Tspan8

On Tuesday, June 21, in the third of four Featured Abstracts being presented by junior investigators during the ISEV 2020 Virtual Annual Meeting (July 20-22) (, Elena Grueso Navarro (photo), a Marie Curie Fellow at TRAIN-EV* at the Institute for Infection Prevention and Hospital Epidemiology, Medical Center-University of Freiburg, Breisgau, German, presented her group’s abstract (FA02) “Nuclear Proteins Are Recruited into Tumor-Derived Extracellular Vesicles Upon Expression of Tetraspanin Tspan8.” Ms. Navarro is a PhD candidate in the laboratory of Irina Nazarenko, PhD, Head, Exosomes & Tumor Group, Institute for Infection Prevention and Hospital Epidemiology, Medical Center-University of Freiburg. In her introduction, Ms. Grueso Navarro noted that tetraspanin 8 (Tspan8) is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in a majority of healthy tissues. When overexpressed, Tspan8 facilitates cell motility, and, in tumor models, it supports invasion and metastasis. In addition, Tspan8 is recruited to the extracellular vesicles (EVs). Previous work in Dr. Nazarenko's laboratory has shown that Tspan8 affects EV content and mediates the EVs’ function in metastasis and angiogenesis. These data suggest that Tspan8 may be a promising therapeutic target in cancer. To learn more about the molecular mechanisms of Tspan8 action on EVs in tumor context, a fibrosarcoma model was established consisting of the parental cell line (HT1080), which is characterized by the absence of endogenous expression of Tspan8 and CD9 tetraspanins, and its derivatives, stably transfected with Tspan8 (HT1080-Tspan8), CD9 (HT1080-CD9), and both tetraspanins together (HT1080-Tspan8/CD9). To investigate the effect of Tspan8 on the EV content, vesicles from conditioned media were purified by size-exclusion chromatography (SEC) and subjected to mass spectroscopy for comparative proteome analysis. The results showed a strong effect of Tspan8 on EV protein cargo. In particular, histones and ribosomal proteins were found among the 20 most-upregulated proteins, which were enriched in the EVs produced by the Tspan8-overexpressing cells. Furthermore, no such effect on the EV proteome was observed in HT1080-CD9-derived vesicles, which supports the hypothesis that the recruitment of nuclear proteins to EVs is a specific effect of tetraspanin Tspan8.

To learn more about the influence of Tspan8 on EV content, a comprehensive RNA analysis was performed. The EV RNA was subjected to next-generation sequencing (NGS) analysis. EVs from HT1080-Tspan8 cells contained a significantly higher amount of total RNA/EV. Subsequently, NGS revealed a difference between HT1080 and HT1080-Tspan8-derived EVs in long RNA content and showed a significant up-regulation of messenger RNA in the EVs upon Tspan8 overexpression.

In conclusion, Ms. Grueso Navarro and colleagues concluded that the data show that the expression of Tspan8 in tumor cells strongly influences the EV cargo in the direction of the upregulation of proteins of nuclear origin and of ribosomal proteins. In fact, this effect seems to be specific for Tspan8 and has not been observed in the upregulation of other tetraspanins. Furthermore, analysis of the RNA content revealed strong differences at the transcriptomic level between the EVs derived from HT1080 and HT1080-Tspan8 cells.

Together, these results indicate the relevance of Tspan8 for the EV content and provide the first hints to the molecular mechanisms of Tspan8 action in cancer.

In addition to having her work selected as one of four ISEV 2020 Featured Abstracts, Ms. Navarro is also a proud ISEV2020 Scholarship Awardee.

In a great honor announced at the ISEV closing ceremony on Wednesday afternoon, Ms. Navarro's work was selected as one of ISEV 2020's Outstanding Contributions to the Advance of Basic Science.

*TRAIN-EV = Training in Extracellular Vesicles: For Benefit in Health and Disease (