title: Role of Plasma Extracellular Vesicles in Lung Cancer Pre-Metastatic Niche Formation
Creators: name: Pontis, Francesca
Publisher: name: The Open University
Year: 2025

description: Lung cancer is one of the most prevalent cancers and, despite numerous advancements in prevention and treatment, still remains the leading cause of cancer-related deaths worldwide. The high mortality rate associated with lung cancer is directly related to the high incidence of regional and distant metastasis. Although the mechanisms of metastasis are not yet fully understood, discoveries made in the past decades have revealed that primary tumours can, even at early stages of the disease, "corrupt" distant microenvironments creating a favourable milieu for cancer cell homing and metastasis initiation known as “pre-metastatic niche” (PMN). Extracellular vesicles and particles (EVPs) have been identified as primary drivers of PMN formation due to their crucial role in intercellular communication. Indeed, as membrane-enclosed vesicles, EVPs from primary tumours can efficiently deliver biomolecules such as proteins and miRNAs to distant organs, providing stability and protection during their travel through the bloodstream. However, in the context of lung cancer, delays in diagnosis and lack of predictive markers of metastasis in early-stage patients pose challenges in such studies. Thus, little is known about lung cancer PMN formation and the role of EVPs in this context. Importantly such studies can allow to discover novel biomarkers which are currently highly needed to develop more effective approaches to prevent and manage metastatic spread in patients with lung cancer.
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For these reasons, the objectives of this PhD project were to investigate the function of plasma EVPs in the context of PMN formation and to identify novel prognostic biomarkers for lung cancer patients in the early stages of the disease. 
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To achieve these goals, plasma EVPs were isolated from a retrospective cohort of 40 early-stage patients who survived for 5 years (ESA) and 40 patients who have died within two years of diagnosis (ESD). In addition, EVPs from the plasma of heavy-smoker cancer-free volunteers (HS) were used as controls.
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EVP characterization and surface markers analysis revealed that alongside common EV-related markers, plasma EVPs markedly express platelet and endothelial markers, revealing the main contributors of circulating EVPs. Moreover, <i>in vitro studies</i> and <i>in vivo</i> biodistribution experiments have unveiled that endothelial cells, along with macrophages (mainly <i>in vivo</i>), are the most avid incorporators of EVPs among stromal cells. Importantly, the obtained results highlighted a prominent role of ESD-EVPs in strikingly induce endothelial cell activation towards a “pro-inflammatory” phenotype (<i>VCAM1</i>, <i>CXCR4</i>, and <i>CXCL1</i> expression and release) as confirmed by <i>in vitro</i> (2D and 3D models) and <i>in vivo</i> experiments. Interestingly, we observed that the upregulation of endothelial CXCL1 and VCAM1 by ESD treatment orchestrated microenvironmental changes by inducing a fibroblast pro-inflammatory response (<i>IL6</i> and <i>CXCL1</i> overexpression) and enhancing neutrophils adhesion, respectively. In addition, the analysis of lung tissues in mice treated with ESD-EVPs confirmed that preconditioning with EVPs led to a greater accumulation of G-MDSC cells and revealed a higher abundance of the Siglec F<sup>+</sup> subpopulation (immunosuppressive) than ESA and HS-EVPs. Dissemination experiments further corroborated the effectiveness of EVPs treatment in PMN establishment as noticed by the higher rate of success of lung cancer cell dissemination and growth in the lungs of mice pre-treated with ESD then in those treated with ESA.
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Moreover, elevated levels of miR-29a, miR-199a, C4A, and SFTPB proteins have been observed in ESD-EVPs compared to ESA and HS-EVPs. Among them, miR-29a and C4A were identified as potential mediators of endothelial activation, as combinatorial treatment of endothelial cells likely recapitulated the ESD-induced phenotype.
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In conclusion, our findings suggest that miR-29a and C4A carried by plasma EVPs drive lung endothelial inflammation, resulting in PMN establishment and metastasis formation. Moreover, EVP-miR-199a, miR-29a, C4A, and SFTPB can be used as predictive biomarkers for patients with early-stage lung cancer.