Project Funding Details

The understanding of tumour development has evolved from a cell-centric to a networking process, with the immune system playing a key role in cancer biology. Neutrophils are found infiltrating the majority of tumors, and can exert both pro-tumoral and anti-tumoral effects. This contradiction could be dependent on aspects of neutrophil biology that have been overlooked for a long time. Recent reports challenged the notion of neutrophils as short-lived and poorly plastic cells, revealing their heterogeneity and plasticity. Thus, the effects of neutrophils in the tumour microenvironment (TME) may depend on their heterogeneity and adaptability to environmental cues. Our preliminary data showed that neutrophils infiltrating 3-Methilcolanthrene (3-MCA)-induced sarcoma exhibited different activation states. We found clusters of neutrophils reprogrammed to immunosuppressive or immunostimulatory states. We aim to investigate the cellular and molecular mechanisms governing neutrophils reprogramming within the TME, and provide suitable target for innovative therapies. Based on our robust preliminiray data and our hypothesis, this proposal is composed of two aims, as follow: • Aim1: the dynamics of neutrophils recruitment, localization, and reprogramming. • Aim2: the role of neutrophils at different stages of tumour development. To fully develop the aims of the present project, we will take advantage of the cutting-edge technologies available in our institute, complemented with genetically engineered mouse models and models of carcinogenesis. Aim1: we will characterize the phenotype of neutrophils at different stages of tumour development by using multiparametric flow-cytometry. Moreover, we will use fluorescent microscopy and spatial mass cytometry to assess the precise localization of neutrophils in the TME, as well as their interactions with other immune cells. We will use the transfer of highly purified neutrophils from Catchup mice to assess their kinetics of recruitment and localization within the TME. In addition, we will be able to associate neutrophil activation state with specific tumour niches. Aim2: we will generate an inducible neutrophil-deficient mouse model by crossing Catchup mice, which express the Cre recombinase under a specific neutrophil promoter (e.g., Ly6g) with B6-iDTR mice. We will challenge this strain with sarcoma models, such as 3-MCA and subcutaneous cell line injection (FS6 and MN-MCA1). Cre-inducible expression of the diphtheria toxin receptor (DTR) following the administration of diphtheria toxin will allow us to produce specific inducible neutrophil depletion. We will deplete neutrophils at different stages of tumour development, and we will evaluate tumour growth and dissect the TME to study the cellular and molecular mechanisms beyond the observed phenotype. The proposal will provide new insights on the dynamics and heterogeneity of neutrophils in the TME, with a focus on the mechanisms responsible for their reprogramming (Aim1). Moreover, we will elucidate the impact of neutrophils at different stages of tumour development (Aim2). The impact of immunotherapies is incomparable with other discoveries in cancer therapy. With this project we aim to provide new insights on neutrophil biology within tumour, as well as promising tools for neutrophils reprogramming in favour of their anti-tumour activity. The final goal is to provide new strategies for the treatment of cancer patients.