Immune Microenvironment Involvement in Neuroendocrine Tumor Behavior and Progression

Neuroendocrine neoplasms (NENs) are rare tumour showing a wide spectrum of clinical behaviours. Therapeutic options available for NETs are rarely curative and mostly palliative, as NETs frequently show resistance to pharmacological therapy. Tumours develop in complex tissue environments, which they depend on. Tumour-associated macrophages (TAMs) are a major cellular component of the tumour microenvironment. Two polarized state of macrophages are described in literature: M1 (anti-tumour promoting effects) and M2 (pro-tumour promoting effects). The importance of immune microenvironment and the significance of macrophages in pancreatic (Pan-) and pulmonary (Lung-) NENs are poorly understood. So, the study of tumour microenvironment could provide new insights into the behaviour of pulmonary and pancreatic NETs, more effective therapeutics strategies that could overcome pharmacological resistance and provide new immune markers. We aimed to characterized TAMs within Pan- and Lung- NEN. Taking advantage of multiparametric flow cytometry analysis, our study highlight that TAMs are present in both Pan- and Lung- NENs. Moreover, the majority of TAMs express M2-like markers as CD206 and CD163, in addition to CCR2 and CXCR2. To assess the effects of M1, M2 macrophages on biological behaviour of NEN, primary cells and NEN cell lines QGP-1 (pancreatic-NEN) and H727 (pulmonary-NEN) were cultured with macrophages conditioned medium (CM). We found out that, only M1 macrophages CM is able to strongly decrease proliferation of primary NEN cells derived from patients. Moreover, data on QGP-1 and H727 cells shown that M1 macrophages have a potent anti-tumor effect able to affect proliferation and tumorigenicity of NEN cell lines. We assessed the capacity of NEN cell lines to secrete different chemokines. We found that our cell lines shown a different behaviour, concerning cytokine production. H727 cells express high levels of CXCR1 and CXCR2 ligands, whereas QGP-1 cells produced high levels of CXCL9 and CCL21. Next, we assessed the effects of NEN cell lines on macrophages and, interestingly, the CM of tumour cell lines promoted the differentiation of macrophages into an M2-like phenotype. Gene expression profile performed on QGP-1 and H727 cells, treated with M1 CM, revealed an enrichment in important pathways knows to be related to tumour suppression. In detail, we found a significant correlation with apoptosis related pathway, p53 pathway and interferon gamma response pathway. These results were matched with MS analysis on CM of M1 macrophages, that revealed an enriched in complement factors, lysozymes and cathepsin. They may play a role in the anti-tumour activity of M1 CM. Finally, we perform the immunophenotype of peripheral blood from NEN patients. I found a decrease in the percentage of lymphocytes, and an increase in the percentage of granulocytes, that correlate with tumour grade or type, in patients bearing a NEN compared to healthy donors.