Dimitrov, Jelena (2024) EVALUATION OF DRIVERS OF METASTATIC DISSEMINATION. [Tesi di dottorato]

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Abstract

Neutrophil extracellular traps (NETs) are complex structures released by activated neutrophils that have been reported to be involved in metastatic dissemination. The aim of our study was to evaluate cancer cell adhesion to NETs and to determine whether cancer cell exposure to NETs can trigger the epithelial-to-mesenchymal transition (EMT), thereby promoting increased migration and invasion of tumor cells. Various cancer cell lines were subjected to a solid-phase adhesion assay using plates coated with NETs, with or without the inclusion of antibodies against the α5β1 Integrin or CCDC25 receptor. After incubation for 1 or 2 hours, the proportion of adherent cells was calculated in relation to the total cell number. To evaluate the induction of EMT, cancer cells were treated with NETs for up to 48 hours and cell lysates were then subjected to western blot for the evaluation of E-cadherin, Vimentin, Snail, Slug, ZEB 1, and TWIST 1 protein levels, as well as Notch 1 and cleaved Notch 1. Moreover, untreated and NET-treated cancer cells underwent migration assays using transwell inserts in 24-well plates, with fetal bovine serum (FBS) serving as a chemoattractant. Adhesion of cancer cells to NET-coated plates ranged between 54% and 93% that was significantly higher than that observed in uncoated plates. The addition of blocking antibodies targeting α5β1 or CCDC25 receptors markedly inhibited cell adhesion to NETs. Prolonged exposure of EGFR-driven lung cancer cell line to NETs led to EMT activation that was shown to be mediated by the upregulation and activation of Notch 1 and confirmed by elevated levels of EMT markers. This transition resulted in a notable loss of epithelial characteristics and a corresponding reduction in the expression of the oncogenic driver. Furthermore, cell migration was significantly enhanced in NET-treated cells compared to untreated controls. Our study showed the dynamic role of NETs that may provide a microenvironment rich in DNA and fibronectin, facilitating the adhesion of many cancer cells at distant locations where prolonged exposure to NETs initiates EMT by activating the Notch 1 signalling pathway, thereby increasing the migratory and invasive potential of cancer cells. Moreover, these findings provided insight into how the immune and inflammatory microenvironment can directly alter the sensitivity of cancer cells to oncogene-targeted agents.

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