A novel method for isolation of tumor infiltrating myeloid-derived suppressor cells from human lung tumor tissue.

The tumor microenvironment comprises different cell subsets including myeloid-derived suppressor cells, which exert intratumoral immunosuppression and favor cancer progression. Isolating tumor-infiltrating myeloid-derived suppressor cells (tMDSCs) from human tumor samples remains a challenge. Current methods such as magnetic bead sorting (MACS) or flow cytometry sorting (FACS) present some drawbacks in terms of purity and viability. Here, we have setup an innovative workflow that combines RosetteSep technology and MACS for isolation of tMDSCs from lung cancer biopsies. To evaluate our Rosette-MACS approach, we compared its performance with MACS and FACS. The isolated cells were characterized by flow cytometry, gene expression analysis and proliferation assays for comparison purposes. The results showed that the Rosette-MACS protocol had the highest yield and purity of tMDSCs (79.64% vs 13.30% with FACS and 0.39% with MACS). Furthermore, the functionality of the isolated tMDSCs was tested not only by upregulation of immunosuppressive genes (e.g. ARG1, IDO1, or PD-L1), but also by their capacity to inhibit CD8(+) T cells proliferation. The combined use of RosetteSep and MACS provides an improved approach for the isolation of functional tMDSCs, which delineates a suitable experimental framework to selectively study the molecular mechanisms underpinning tMDSCs-derived immunosuppression in the TME.