In vitro differentiation of M1, M2, and TAM-like macrophages from healthy donor PBMC
To  understand the immunological role of different macrophage populations in the tumor, we generated transcriptional profiles of macrophages differentiated from PBMC-derived monocytes in vitro under three conditions to compare with: M1-like macrophages were developed in vitro through polarization of human monocytes by IFNg and TLR4 stimulation. This population is considered to have anti-tumor activities; releasing proinflammatory cytokines, performing phagocytosis, and possessing antigen cross presentation abilities. Second, M2-like macrophages were developed in vitro through intensive IL4 stimulation of monocytes. M2 macrophages are known to promote many pro-tumorigenic processes such as angiogenesis, immune suppression, hypoxia induction, tumor cell proliferation,  metastasis. Lastly, we deployed a third population that represents a better in vitro model for tumor associated macrophages (TAM), developed under conditions better resembling the TME.Monoctyes derived from two individual patient donors were cultured separately under four (4) different conditions in vitro, for a total of eight (8) samples to be processed for bulk RNA sequencing. Overall design: Monoctyes derived from two individual patient donors were cultured separately under four (4) different conditions in vitro, for a total of eight (8) samples to be processed for bulk RNA sequencing