Targeting chemokine receptors to promote anti-tumor responses of neutrophils

Neutrophils represent a relevant component of the tumor microenvironment, and their presence in tumor tissue and high neutrophilia have been associated with poor prognosis in different oncological contexts, like high grade gliomas (HGGs). However, neutrophils are not a homogeneous population, and they can play a dichotomous role in cancer being both anti- and pro-tumoral. Indeed, tumor-derived cytokines induce the appearance in the blood of immature neutrophils with immunosuppressive activities, and neutrophils with an “aged” phenotype still able to respond to inflammatory stimuli. It is evident that understanding neutrophil heterogeneity and how to harness neutrophil antitumoral potential represent important therapeutic options to improve current cancer treatments and patient clinical outcomes. The chemokine system regulates neutrophil trafficking from the bone marrow to the bloodstream and to the tumor tissue. Beyond their chemotactic role, chemokines were reported to have effects also in neutrophil effector functions such as ROS production and NETs release. However, the role of chemokines and their receptors in neutrophil heterogeneity and effector activities in the tumor context is not clearly defined. Thus, the first aim of this thesis is to study the role of chemokine receptors in the differentiation and activation of neutrophils both in vitro and in vivo, to test if chemokine receptors could be used to track or modulate neutrophil subpopulations with different effector functions. We focused our attention on the CXCL8-CXCR2 axis, which is highly relevant for neutrophil biology in tumors. In vitro stimulation of isolated human neutrophils with CXCL8 resulted in a unique neutrophil phenotype, different from classically activated or N1 neutrophils with characteristics of non-activated mature neutrophils. Moreover, in an in vivo model of metastatic melanoma, we found that mice treated with a CXCR2 antagonist but not with a CXCR4 antagonist were protected from metastases compared to control mice. Unexpectedly, we found an increased number of circulating neutrophils in anti-CXCR2 treated mice compared to anti-CXCR4 treated and control mice. These results indicated that the interference with CXCL8-CXCR2 axis in a metastasis model does not reduce neutrophil mobilization while resulting in metastasis protection. Further analyses on neutrophil activation and maturation state and on other immune cells having antimetastatic roles will be necessary to understand the mechanism of anti-CXCR2 protection from metastases. The second aim of the thesis was to study the predictive potential of circulating and tumor-associated neutrophil subpopulations ex vivo in patients with HGGs. Single cell analysis of tumor samples of HGG patients revealed that patients with worse prognosis (IDH1-WT), compared to IDH1-mut and healthy samples, showed a higher leukocyte infiltration. Because it was not possible to clearly identify neutrophils, we performed a FACS analysis on tumor lesions and blood samples. We found that IDH1-WT patients have a tendendy to have more infiltrating neutrophils and a significantly higher neutrophilia compared to IDH-mut patients and healthy samples. However, we did not find any significant correlation between the number of pre-surgery neutrophils and patient progression free survival (PFS). Unexpectedly, analysing circulating neutrophil subpopulations, we found that the number of pre-surgical immature neutrophils correlated positively with PFS. We obtained the same result analysing circulating neutrophils in a cohort of HGG patients undergoing immunotherapy. In order to deeper investigate the role of neutrophil subsets in this tumor context, we set up an in vivo model of glioma by orthotopic injection of GL261 RedFluc neurospheres.