Neutrophils represent the most abundant cell type of leukocytes in the human blood and have been considered a vital player in the innate immune system and the first line of defense against invading pathogens. Recently, several studies showed that neutrophils play an active role in the immune response during cancer development. They exhibited both pro-oncogenic and anti-tumor activities under the influence of various mediators in the tumor microenvironment. Neutrophils can be divided into several subpopulations, thus contradicting the traditional concept of neutrophils as a homogeneous population with a specific function in the innate immunity and opening new horizons for cancer therapy. Despite the promising achievements in this field, a full understanding of tumor–neutrophil interplay is currently lacking. In this review, we try to summarize the current view on neutrophil heterogeneity in cancer, discuss the different communication pathways between tumors and neutrophils, and focus on the implementation of these new findings to develop promising neutrophil-based cancer therapies.

Many studies have reported an increase in the level of circulating cell-free DNA (cfDNA) in the blood of patients with cancer. cfDNA mainly comes from tumor cells and, therefore, carries features of its genomic profile. Moreover, tumor-derived cfDNA can act like oncoviruses, entering the cells of vulnerable organs, transforming them and forming metastatic nodes. Another source of cfDNA is immune cells, including neutrophils that generate neutrophil extracellular traps (NETs). Despite the potential eliminative effect of NETs on tumors, in some cases, their excessive generation provokes tumor growth as well as invasion. Considering both possible pathological contributions of cfDNA, as an agent of oncotransformation and the main component of NETs, the study of deoxyribonucleases (DNases) as anticancer and antimetastatic agents is important and promising. This review considers the pathological role of cfDNA in cancer development and the role of DNases as agents to prevent and/or prohibit tumor progression and the development of metastases.

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme^®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme^® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme^® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme^®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.

Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils.

Currently, a significant increase in the levels of circulating cell-free DNA (cfDNA) in the blood of patients is considered as a generally recognized marker of the development of oncological diseases. Although the tumor-associated cfDNA has been well studied, its biological functions remain unclear. In this work, we investigated the effect of cfDNA isolated from the blood serum of the mice with B16-F10 metastatic melanoma on the properties of the B16-F10 melanoma cells in vitro . It was found that the profile of cfDNA isolated from the blood serum of mice with melanoma differs significantly from the cfDNA isolated from the blood serum of healthy mice, and is similar to the genomic DNA of B16 cells with regards to abundance of oncogenes and mobile genetic elements (MGE). It was shown that the cfDNA of mice with melanoma penetrated into B16 cells, resulting in the increase in abundance of oncogenes and MGE fragments, and caused 5-fold increase of the mRNA level of the secreted DNase Dnase1l3 and a slight increase of the mRNA level of the Jun , Fos , Ras , and Myc oncogenes. cfDNA of the healthy mice caused increase of the mRNA level of intracellular regulatory DNase EndoG and 4-fold increase of the mRNA level of Fos and Ras oncogenes, which are well-known triggers of a large number of signal cascades, from apoptosis inhibition to increased tumor cell proliferation. Thus, it is obvious that the circulating cfDNA of tumor origin is able to penetrate into the cells and, despite the fact that no changes were found in the level of viability and migration activity of the tumor cells, cfDNA, even with a single exposure, can cause changes at the cellular level that increase oncogenicity of the recipient cells.

It is widely postulated that the majority of pathologically elevated extracellular or cell-free DNA (cfDNA) in cancer originates from tumor cells; however, evidence has emerged regarding the significant contributions of other cells from the tumor microenvironment. Here, the effect of cfDNA originating from murine B16 melanoma cells and L929 fibroblasts on B16 cells was investigated. It was found that cfDNAL929 increased the viability and migration properties of B16 cells in vitro and their invasiveness in vivo. In contrast, cfDNAB16 exhibited a negative effect on B16 cells, reducing their viability and migration in vitro, which in vivo led to decreased tumor size and metastasis number. It was shown that cell treatment with both cfDNAs resulted in an increase in the expression of genes encoding DNases and the oncogenes Braf, Kras, and Myc. cfDNAL929-treated cells were shown to experience oxidative stress. Gene expression changes in the case of cfDNAB16 treatment are well correlated with the observed decrease in proliferation and migration of B16 cells. The obtained data may indicate the possible involvement of fibroblast DNA in the tumor microenvironment in tumor progression and, potentially, in the formation of new tumor foci due to the transformation of normal cells.