Table 2.

NET formation inhibitors

Substance	Manuscript	Experimental model	Findings	Comments
Anthracyclines (e.g. epirubicin, daunorubicin, doxorubicin and idarubicin)	Khan et al. [74]	Human neutrophils, in vitro	Both NOX-dependent and NOX-independent NET expression were blocked.	Anthracyclines’ effect may be mediated by alteration of transcription initiation.
Anti-GCSF antibodies	Park et al. [42, 75]	Murine model. 4T1 breast cancer cells	Reduced cancer cell–induced NET expression and metastases.	In humans, these could be difficult as GCSF is commonly used as a treatment of neutropenic sepsis in cancer patients [42].
Anti-HMGB1 antibodies	Tohme et al. [26]	In vitro. MC 38 colorectal cancer cells	NET-mediated tumour cell proliferation, invasion and migration were significantly decreased (p < 0.001).	Leads to MAP kinase pathway inhibition
Antithrombin III	Ishikawa et al. [76]	Murine model of LPS-induced endotoxemia	Reduced mortality, lung neutrophil infiltration, expression of NETs, HMGB1 protein and CXCL1 and 2	In addition to the anticoagulation effects, antithrombin III has also anti-inflammatory effects.
	Jung et al. [27•]	In vitro. AsPC-1 pancreatic cancer cells	Significant inhibition of AsPC-1-induced NET formation
COX inhibitors	Tilgner et al. [69]	In vivo, LPS-induced acute lung injury model in C57Bl/6 mice	Aspirin significantly decreased lung and plasma NET levels.	Aspirin decreased neutrophil infiltration and NET formation via CCL5 chemokine and PAD4 inhibition.
Diethylcarbamazine	Segoviano-Ramirez et al. [77]	In vitro. polymorphonuclear cells (PMNs) from healthy and type 2 diabetes mellitus patients	NET formation was significantly inhibited both in healthy (p ≤ 0.000001) and DM2 (p ≤ 0.000477) volunteers.	Although a drug used as an anti-parasitic, this could be useful in NET expression modulation in diabetic wound healing and cancer potentially.
DNase	Jung et al. [27•]	In vitro, AsPC-1 pancreatic cancer cells	Significant inhibition of AsPC-1 NETs promoted cancer cell migration.	Among all other modulators, DNase has the largest use in various experimental models and is already in clinical use for CF treatment. It may likely be among the first drugs to be used in cancer for outcome control. Preliminary in vitro and animal results suggest DNAse reduces NET formation, with effects on tumour cell adhesion, tumour burden and metastasis [26, 42].
	Tohme et al. [26]	In vitro and a murine model. MC 38 colorectal cancer cells	Significant inhibition of tumour adhesion, the development and growth of metastatic disease
	Park et al. [42]	Murine model. 4T1 breast cancer cells	DNase-coated nanoparticles decreased lung metastatic burden.
	Kolaczkowska et al. [78]	Murine model of methicillin-resistant Staphylococcus aureus bacteraemia. PAD4 and NE-negative mice	DNase failed to remove most histones form the vessel wall and only partially reduced the endothelial injury. Inhibition of NET formation (in murine models of PAD4 and NE deficiency) also prevented collateral host tissue damage.
Gasdermin D inhibitor LDC7559	Sollberger et al. [79]	In vitro, human neutrophils	Gasdermine D is required for NET expression and acts upstream of NE. LDC7559 reduced histone H3 protein processing.	Gasdermin D is a pore-forming protein involved in the release of granular proteins required for NET expression and facilitates NE translocation to the nucleus. Also forms membrane pores releasing the NETs in the extracellular space.
Heparin and polysialic acid (PSA)	Jung et al. [27•]	In vitro, AsPC-1 pancreatic cancer cells	Blocked histone-induced angiogenesis	These two substances have in common a highly negative electric charge which binds to positively charged histones, leading to blockage of NET-induced cancer cell migration and angiogenesis [27•, 80].
HMG-CoA reductase inhibitors	Sapey et al. [81]	In vitro. neutrophils from patients receiving high-dose simvastatin for community-acquired pneumonia	Significant reduction in NET expression and NE activity on day 4. No difference in mortality and length of stay	The result of these studies looking at the NETs role in response to bacteria is very different. Keeping in mind the different experimental models, translation of anti-NET therapies from bench to bedside may prove difficult and may not be associated with a clinical effect. This could be the case in cancer too.
	Chow et al. [82]	In vitro. Human and mice PMNs, cultured with various statins: mevastatin, simvastatin, lovastatin and fluvastatin. The NET formation was quantified in response to bacterial exposure.	Statins boosted the production of anti-bacterial DNA-based extracellular traps by human and murine neutrophil (p < 0.005)
Lidocaine	Galos et al. [67••]	Patients undergoing primary breast cancer resection	Lidocaine infusion decreased expression of citH3 and MMP-3.	This study offers the premises for a large trial regarding the impact of lidocaine on the metastasis.
NADPH oxidase 2 (NOX2) inhibitors (e.g. apocyanin)	Park et al. [42]	In vitro. Murine model. 4T1 breast cancer cells	NOX2 inhibition reduced NET formation and neutrophil-promoted cancer cell invasion.	NADPH oxidase is a critical enzyme as shown already in the NET production process. Although inhibitors against it are available and represent a potential tool for blocking NET production, it is not feasible as this enzyme is involved in critical processes like bacterial killing, phagocytosis and degranulation [42].
PAD4 inhibition (e.g. YW4-03, Cl-amidine)	Tohme et al. [26]	In vitro and a murine model. MC 38 colorectal cancer cells. PAD4-deficient mice	In vitro, PAD4 inhibitors or the use of PAD4-deficient neutrophils block the adhesion of tumour cells to a monolayer of NETs expressing neutrophils. Significantly reduced the tumour cell growth, p < 0.0001 Levels of cit-H3 in the tumours after 3 weeks were absent in PAD4−/− mice and mice receiving YW4-03.	PAD-4 inhibition has been associated with preventing NET formation in animal models, but its effect on metastasis will be difficult to predict due to the short half-lives of the current commercially available inhibitors [42].
	Park et al. [42]	Murine model. 4T1 breast cancer cells	It decreased NET formation (p = 0.02). No effect on tumour cell migration
PGE1	Jung et al. [27•]	In vitro, AsPC-1 pancreatic cancer cells	Inhibited cancer cell–induced NET formation, by decreasing the intracellular calcium content	PGE 1 has been used clinically for neonatal duct–dependent disease, sexual dysfunction, critical limb ischemia due to its anti-inflammatory, vasodilative and anti-platelet effects [83].
Ph	De Souza et al. [84]	In vitro. Human neutrophils	Raising the pH promotes intracellular calcium influx which leads to increases PAD4 activity, citrullination of histone, histone cleavage and NET formation.	Both extra and intracellular alkaline Ph have been linked with cancer promotion [85]. Also, Ph-sensitive channels could become a therapeutic target in some cancers [86]. The alkaline pH modulates the NET formation as enzymes like PAD4 and NE prefer an alkaline environment and processes like mitochondrial ROS and PAD4-mediate citrullination are augmented by alkaline pH with an impact on both NOX-dependent and NOX-independent processes [84, 87].