Immune evasion hallmark has grabbed wide attention in cancer progression on the clinical level. Accordingly, innate and adaptive immune cells isolation and manipulation is essential in order to assess their activity and role in the tumor microenvironment (TME). This could open a gate toward a personalized therapy by a simple aspiration of blood sample from patients. Here, we describe the isolation of peripheral blood mononuclear cells (PBMCs) using Ficoll plus media in order to achieve the highest yield of immune cells that can be further processed and used in isolation of specific immune cells such as macrophages and cytotoxic T cells (CD8+ cells). Among the highly metastatic macrophages are the M2. This protocol describes the optimized techniques to isolate monocytes from whole blood, differentiate them into M2. This is followed by genetic and epigenetic (using synthetic nucleotides of noncoding RNAs) manipulation of these isolated immune cells in a tumor culture media, in addition to measurement of released cytokines using specific ELISA kit. In the last decade, new groups of noncoding RNAs have been emerged which are microRNAs and long noncoding RNAs. First, they were known as "junk DNA" with unknown regulatory functions. Despite the limited knowledge of these molecules, basic expression profiling is proving to be clinically relevant to cancer diagnosis, metastasis, and prognosis. Here, we describe methods used in molecular biology to assess the epigenetic expression of ncRNAs and their impact on other messenger RNA transcripts in M2 macrophages that could serve as future biomarkers in the context of tumor biology and metastasis or could open a gate in the treatment of cancer.
Keywords: Cytotoxic T cells; Macrophages differentiation; Macrophages isolation; PBMCs isolation; Transfection; cDNA; qRT-PCR.