Introduction: An increased release of neutrophil extracellular traps (NETs) through NETosis, occurs in cancer. This enhances tumor growth and metastasis, which may be mediated by impaired NET degradation. However, this has never been evidenced in bladder cancer (BC). To evaluate the increase in NETosis in BC patients, to ascertain whether it is mediated by plasma DNaseI alterations and to in vitro explore therapeutic interventions. Methods: NETs markers were measured in plasma samples of 73 BC patients and 64 controls. Plasma DNaseI activity was measured with the SRED assay. To evaluate the ability of plasma to degrade NETs, neutrophils were isolated from a healthy donor, seeded, and activated for 4h with phorbol myristate acetate (PMA) to prompt NETosis. NETs were incubated for 6h with 5 µl plasma from each participant and fluorescence was measured after staining with SytoxGreen. In parallel, BC plasma was supplemented with the recombinant human DNaseI Dornase Alfa (Pulmozyme, Roche), or vehicle (PBS 1X + 0.1%BSA) to restore their DNaseI deficiency. We evaluated the presence of NETosis in tumor tissue by immunofluorescence (DNA, citrullinatedH3 and elastase). Statistical analysis was conducted with Graphpad (v.8.0.1). Results: BC patients had significantly increased NETs markers (P <0.0001) and lower DNaseI activity (P <0.0001) in plasma compared to controls (Figure 1). In vitro NET degradation was impaired when incubated with BC plasma compared to control plasma (P <0.0001) (Figure 2). Addition of DNaseI restored NET degradation up to the level of healthy controls (P <0.0001) (Figure 2). NETosis occurs in BC tissue, more profusely in muscle-invasive BC. Conclusions: BC patients have increased NETosis in the tumor microenvironment and plasma, in part mediated by a reduced DNaseI activity. In vitro addition of Dornase Alfa, an approved treatment for cystic fibrosis, restores NET degradation in BC patients, thus becoming a potential therapeutic tool to reduce metastasis. SOURCE OF Funding: ISCIII-FEDER (PI17/00495, PI20/00075, FI21/00171), GVA (CIACIF/2021/192) and SETH.
