Fig 2: Intraperitoneal PMN-MDSCs substantially increase with PD progression. (A) Female C57BL/6J mice (n=6) were intraperitoneally (i.p.) or subcutaneously (s.c.) inoculated with 5×105 MC38 colon cancer cells and sacrificed to collect peritoneal lavage, spleens, and peripheral blood on Days 6, 13 and 20 as indicated. (B) Representative flow cytometric data of intraperitoneal immune cell subsets. The cells were defined as follows: CD11b+F4/80−Ly6ChighLy6G− for monocytic myeloid-derived suppressor cells (M-MDSCs), CD11b+F4/80−CD244+Ly6CmidLy6G+ for polymorphonuclear (PMN)-MDSCs, CD11b+F4/80−CD244−Ly6CmidLy6G+ for neutrophils, CD11b+F4/80+ for macrophages, CD3+CD4+ for CD4 T cells, CD3+CD8+ for CD8 T cells, CD3+CD4+CD25+CD127− for regulatory T cells (Tregs), CD19+CD11b+ for B1 cells, CD19+CD11b− for B2 cells, and CD19+CD1d+CD5+ for B10 cells. (C) Time-course enumeration of the immune cell numbers identified. (D) Time-course enumeration of the number of MDSCs in the peritoneal cavity, spleen, and peripheral blood in the MC38-based peritoneal dissemination (PD) and subcutaneously (SC) inoculated models. Female BALB/c mice (n=4) were intraperitoneally inoculated with 1×105 CT26 colon cancer cells and sacrificed to collect peritoneal lavage and peripheral blood on Days 6, 13 and 20. (E) Representative flow cytometric data of intraperitoneal CD11b+Ly6G+ cells and M-MDSCs. (F) Time-course enumeration of their frequencies in the peritoneal cavity and the peripheral blood. Values are expressed as mean values with standard errors. One-way ANOVA with Holm's post-hoc test was performed. *P<0.05, **P<0.01.

Fig 3: Ly6G+CD244+ cells function as PMN-MDSCs. Spleens were harvested from the MC38-based subcutaneous inoculation (SC) model at 20–30 days after tumor inoculation. Splenocytes were then purified. Ly6G+CD244+ and Ly6G+CD244- cells were further isolated from the splenocytes by a FACS Aria instrument. (A) The gene expression levels of Arginase-1 (ARG1), nitric oxide synthase 2 (NOS2), and myeloperoxidase (MPO) in Ly6G+CD244+ and Ly6G+CD244- cells were measured by quantitative real-time (q)PCR. GAPDH was used to normalize gene expression data. CFSE-labeled OT-1 splenocytes (5×105) were co-cultured with Ly6G+CD244+ or Ly6G+CD244- cells at indicated ratios with SIINFEKL peptides. CFSE fluorescence of OT-1 CD8+ T cells was measured by flow cytometry. (B) Proliferation rates of OT-1 CD8+ T cells, normalized with Ly6G+CD244- cells. (C) The total number of OT-1 CD8+ T cells. Values are expressed as mean values with standard errors. Student's t-test was performed for two group comparisons. *P<0.05, **P<0.01. PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells.