Figure 4

Molecular characteristics of CRC cells and the induction of MDSCs via SW480 or SW620 cells in vitro. (A) The expression of VEGF, G-CSF, IL-6, IL-37, CD73, iNOS, IDO, and COX2 in in SW480 and SW620 cells and in tumor and paraneoplastic tissues from 5 CRC patients was measured using quantitative RT-PCR. GAPDH expression was included as a control. (B) CD33⁺ cells were isolated from healthy PBMCs using human CD33 MicroBeads and were co-cultured with SW480 or SW620 cells. The representative dot plots and statistical graph show the proportion of HLA-DR⁻CD33⁺CD11b⁺ MDSCs induced from CD33⁺ cells by co-culture with SW480 or SW620 cells in a Transwell system for 48 hours. The CD33⁺ cells in medium alone were included as a control. (C) A representative cytospin of HLA-DR-CD33 + CD11b + MDSCs stained with Wright-Giemsa and identified by the mononuclear or polymorphonuclear cell nuclear staining (purple) using light microscopy (20 × 0.30 objective magnification) (Nikon Tokyo, Japan). (D) The phenotypes of the tumor-induced MDSCs were analyzed with flow cytometry using multiple anti-human mAbs against CD14, CD15, CD66b, CD39, CD73, CXCR4, CD117, CD34, Arg-1, iNOS, PD-L1 and ROS, and the grey curve represents autofluorescence as a negative control. Representative histograms are shown. (E) The mRNA levels of TGF-β, IDO, IL-10, IFN-γ, iNOS, Arg-1, and NOX2 in the T-MDSC by CRC cells, and CD33⁺ cells were detected using quantitative RT-PCR; GAPDH was included as control, and one of 5 experiments is shown here. T-MDSC, tumor-induced MDSCs by CRC cells.