NETosis in cancer: a critical analysis of the impact of cancer on neutrophil extracellular trap (NET) release in lung cancer patients vs. mice.

Neutrophils play a major role in tumor biology. Among other functions, neutrophils can release extracellular traps (NETs), mesh-like structures of decondensed chromatin fibers, in a process termed NETosis. Originally characterized as an antimicrobial mechanism, NETosis has been described in cancer, but cancer-related predisposition is not clear. In the current study, we investigated the predisposition of circulating neutrophils to release NETs in lung cancer and the impact of G-CSF on this function, comparing circulating neutrophils isolated from cancer patients to the LLC and AB12 mouse models. We find that neutrophils from both healthy donors and cancer patients display high NETotic potential, with 30-60% of cells undergoing NETosis upon PMA stimulation. In contrast, neutrophils isolated from tumor-bearing mice displayed only 4-5% NETotic cells, though significantly higher than naive controls (1-2%). Despite differential mechanisms of activation described, Ionomycin and PMA mainly triggered suicidal rather than vital NETosis. G-CSF secreting tumors did not increase NETotic rates in murine neutrophils, and direct G-CSF stimulation did not promote their NET release. In contrast, human neutrophils strongly responded to G-CSF stimulation resulting also in a higher response to PMA + G-CSF stimulation. Our data show clear differences in NETotic potentials between human and murine neutrophils. We do not find a predisposition of neutrophils to release NETs in lung cancer patients compared to healthy controls, whereas cancer may modulate neutrophils' NETotic potential in mice. G-CSF secreted from tumors differentially affects murine and human NETosis in cancer. These important differences should be considered in future studies of NETosis in cancer.

The Bilateral Interplay between Cancer Immunotherapies and Neutrophils' Phenotypes and Sub-Populations.

Immunotherapy has become a leading modality for the treatment of cancer, but despite its increasing success, a substantial number of patients do not benefit from it. Cancer-related neutrophils have become, in recent years, a subject of growing interest. Distinct sub-populations of neutrophils have been identified at advanced stages of cancer. In this study, we aimed to evaluate the role of neutrophils in mediating the efficacy of immune checkpoint inhibitors (ICI) treatments (α-PD-1/PD-L1), by assessing lung tumor models in mice. We found that G-CSF overexpression by the tumor significantly potentiates the efficacy of ICI, whereas neutrophils' depletion abrogated their responses. Adoptive transfer of circulating normal-density neutrophils (NDN) resulted in significantly reduced tumor growth, whereas low-density neutrophils (LDN) had no effect. We next investigated the effect of ICI on neutrophils' functions. Following α-PD-L1 treatment, NDN displayed increased ROS production and increased cytotoxicity toward tumor cells but decreased degranulation. Together, our results suggest that neutrophils are important mediators of the ICI treatments and that mainly NDN are modulated following α-PD-L1 treatment. This research provides a better understanding of the function of neutrophils following immunotherapies and their impact on the efficacy of immunotherapy, supporting better understanding and future improvement of currently available treatments.

Tumor-Associated Neutrophils Drive B-cell Recruitment and Their Differentiation to Plasma Cells.

A major mechanism through which neutrophils have been suggested to modulate tumor progression involves the interaction and subsequent modulation of other infiltrating immune cells. B cells have been found to infiltrate various cancer types and play a role in tumor immunity, offering new immunotherapy opportunities. Nevertheless, the specific impact of tumor-associated neutrophils (TAN) on B cells has largely been overlooked. In the current study, we aimed to characterize the role of TANs in the recruitment and modulation of B cells in the tumor microenvironment (TME). We showed that TANs actively participate in the recruitment of B cells to the TME and identified TNFα as the major cytokine mediating B-cell chemotaxis by TANs. The recruitment of CD45+B220+CD138- splenic B cells by TANs in vitro resulted in B-cell phenotypic modulation, with 68.6% ± 2.1% of the total migrated B cells displaying a CD45-B220+CD138+ phenotype, which is typical for plasma cells. This phenotype mirrored the large proportion (54.0% ± 6.1%) of CD45-B220+CD138+ intratumoral B cells (i.e., plasma cells) in Lewis lung carcinoma tumors. We next confirmed that the differentiation of CD45+B220+CD138- B cells to functionally active CD45-B220+CD138+ plasma cells required contact with TANs, was independent of T cells, and resulted in IgG production. We further identified membranal B-cell activating factor (BAFF) on TANs as a potential contact mechanism mediating B-cell differentiation, as blocking BAFF-receptor (BAFF-R) significantly reduced IgG production by 20%. Our study, therefore, demonstrates that TANs drive the recruitment and modulation of B cells into plasma cells in the TME, hence opening new avenues in the targeting of the immune system in cancer.