Assessment of lipid load in tumor-infiltrating Tregs by flow cytometry.

Regulatory T cells (Tregs), expressing the transcription factor Foxp3, are defined as immunosuppressive cells able to modulate a variety of immune cells in order to avoid unwanted and excessive immune responses; however, in the tumor context, Treg function contribute to inhibit immune surveillance, thus promoting cancer progression. In tumor microenvironment, where the availability of metabolic resources is strongly limited, Tregs are expanded and may exploit different metabolic routes to achieve a metabolic advantage, prevailing over effector cells. In this context an important role of lipid metabolism has been described thanks to the possibility to evaluate the intracellular lipid content selectively in tumor-infiltrating Tregs (TUM-Tregs). Taking into account the heterogeneous and complex build of tumor mass, we set-up a combined protocol that optimizes tumor-infiltrating lymphocytes (TIL) extraction from the tissue through a Percoll density gradient, and assesses ex vivo the lipid load in whole TUM-Treg population, evaluating by flow cytometry the incorporation of an intensely fluorescent lipophilic fluorophore able to specifically stain neutral lipids. This method provides an important advantage compared to the traditional technique based on microscopy, whose lipid level evaluation is limited to a tissue section, and hence may not be representative of the entire population.