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Head and neck squamous cell carcinomas (HNSCCs) are aggressive tumours since they are associated with poor treatment responsiveness and poor prognosis, only 50% of patients are alive 5 years after diagnosis. The adaptive immune response plays a significant role in the tumorigenesis. In fact, immune cells are major components of the tumor microenvironment (TME), particularly, the tumour-associated macrophages (TAMs) which are involved in many tumorigenic steps. The macrophages are basically categorised into pro-inflammatory M1 versus anti-inflammatory M2, related to TAMs. The latter are an interesting target to fight cancer. A lot of strategies aimed to induce a switch to macrophage phenotype in order to produce an anti-tumoral TME. The methodology of macrophages polarization is highly controversial in the literature, so we would like to standardize this step. After all, we would like to apply this protocol to study the dual influence of these macrophages in HNSCCs cell lines and also in order to switch TAMs immunosuppressive in anti-tumor phenotype.

First, we carried out and validated a monocyte-to-macrophage polarization protocol to generate some macrophage subtypes (M0, M1 and M2). M0 were generated through exposure of THP1 monocytes to different concentrations (25 or 100 ng/ml) of phorbol 12-myristate-13 acetate (PMA) during 24h. Then, M0 were differentiated in M1 with lipopolysaccharides (LPS) and IFN-γ or in M2 with IL-4 and IL-13 human interleukins. The different phenotypes were confirmed by the identification of some characteristic markers by immunofluorescence (CD14, CD36, CD68, CD86, CD206, NRF2, NF-κB), RTqPCR (CD68, CD80, SOCS1, SOCS3, IL-12, PD-L1, CCL2, CD206, KGA, SLC1A5, NOX2, SOD2, NRF2) and FACS (CD14, CD68, CD80, CD163, CD206). Moreover, we evaluate the stability of the different phenotypes over 72h. We would like to switch M2 phenotype to M1 by targeting metabolism using CB839 and ROS by using PRIMA-^1MET. We will also irradiate M2 with x-ray and expose them with Fe₂O₃ nanoparticles.

We demonstrated that the choice of PMA concentration has a critical impact on the molecular signature and the gene expression of the different subtypes induced. Indeed, the use of 25ng/ml concentration seems to be more appropriate than 100ng/ml to polarize monocyte to M1 macrophages. But, M2 phenotype seems to be more specific with higher concentration (100ng/ml). Polarized-macrophages, M1 and M2, seem to be stable in time. 

With this study we highlight that appropriated polarization conditions are crucial to obtain some distinct immune cell lines for in vitro study. In the oncology field, targeting specifically these TAMs will be a promising therapeutic approach based on the continuum plasticity and flexibility between macrophages phenotypes.