Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

Abstract

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer [1][2][3][4][5][6][7] . In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens 7 . It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to naïve counterparts 7-10 . However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression 11,12 . Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to naïve MDSC 13 . MDSC percentages are significantly less in lymphoid compartments of naïve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1 + leukocytes from naïve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1 + leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1 + leukocytes from spleens of naïve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas naïve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from naïve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1 + leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1 + enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (naïve vs. tumor-bearing) in in vivo and in vitro assays.

Apigenin: Selective CK2 inhibitor increases Ikaros expression and improves T cell homeostasis and function in murine pancreatic cancer

“…In addition, we have previously published that other immunosuppressive cells such as myeloid derived suppressor cells (MDSC) are expanded in TB mice [ 42 ]. MDSC are immature macrophages, dendritic cells and granulocytes [ 58 ]. API reduction in MDSC percentages may be a result of maturation or differentiation of these immature cells, thus producing mature DC, macrophages and other APCs, which could also account for the increased allogeneic immune responses.…”

Apigenin Increases SHIP-1 Expression, Promotes Tumoricidal Macrophages and Anti-Tumor Immune Responses in Murine Pancreatic Cancer

“…All pancreatic tumors were digested with Collagenase, type IV (Sigma-Aldrich), DNase, type IV (Sigma-Aldrich) and Hyaluronidase, Type V (Sigma-Aldrich) in Hank's Balanced Salt Solution (without CaCl 2 or MgCl 2 ) for 1 h [59]. Single cell suspensions were counted and then resuspended in 3% FBS/PBS and surface stained with fluorescent antibodies against murine T cell and myeloid cell surface markers, as previously described [60]. MDSC-TAM subsets were detected with anti-mouse CD11b-APC, anti-mouse Ly6C-PE/Cy7, anti-mouse Ly6G-PerCP, anti-mouse F4/80-BV650, anti-mouse CD86-AlexaFluor700 and anti-mouse CD206-FITC (Biolegend, San Diego, CA, USA).…”

HMGB1 promotes myeloid-derived suppressor cells and renal cell carcinoma immune escape

“…Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous cells derived from bone marrow that have a significant inhibitory effect on immune cell responses. Gradually, the key role of MDSCs in the development of tumors had also been confirmed [ 9 , 10 ]. Many studies showed that MDSCs was involved in tumor immune escape and promoted tumor progression [ 11 , 12 ].…”