Figure 2.

Multiple impacts of sCRT/39-272 on myeloid derived suppressor cells (MDSCs). (A) Groups of female C57BL/6 mice (n = 6) were s.c. injected with B16-EGFP or B16-CRT cells (5 × 10⁶/100 μl/mouse) and sacrificed 21 days postinoculation for their solid tumors. Tumor tissues were made into 5 μM frozen section for immune-fluorescence staining using Alexafluor 647-labeled anti-Gr1 Abs (red) and DAPI (blue). Images were acquired by laser-scanning confocal microscopy. Arrows indicate Gr1⁺ MDSCs. (B) B16-CRT and B16-EGFP tumor tissues were cut into small pieces and digested with collagenase and DNase digestion. Single cells were filtered through 40-µm nylon mesh after further mechanically dissociated with a 10-ml pipette and followed by staining with fluorescence labeled anti-CD11b and anti-Gr1 Abs. Percentage of Gr1-positive MDSCs among the tumor cells was analyzed by FACS. (C) For in vitro migration assays, MDSCs, purified from the spleens of tumor-bearing mice using biotin labeled anti-Ly6G-coupled microbeads, were placed in the top chamber of a transwell, recombinant sCRT/39-272 or recombinant enhanced green fluorescence protein (rEGFP) was placed in the bottom chamber. After 3 h, transwell filters were fixed with 4% paraformaldehyde for 30 min, followed by wiping away the remaining cells in the top chamber and then DAPI staining, cells was visualized by fluorescence microscope. Representative micrographs are shown on the left and statistic result of 6 different vision cells on the right. (D) MDSCs purified from tumor-bearing mice were incubated with sCRT/39-272 or rEGFP for 20 h, followed stained with fluorescence-labeled Annexin-V and 7-AAD and detected by FACS. The results were replicated for three times with consistent trend, representative results of the three repeating experiments are shown. **p < 0.01.