MUC1/sec blocks ROS production in MDSCs. (A) Splenic MDSCs from DA-3/TM tumor-bearing mice were cultured in vitro for 18 hours in the presence of IL-4, 20% DA-3/TM CM, DA-3/sec CM, DA-3/sec CM separated into MUC1/sec-containing fraction (sec[> 100 K]), and/or DA-3/sec CM lacking MUC1/sec (sec[< 100 K]). NOHA was used in cultures as a control for inhibition of ROS production by MDSCs. ROS was detected by treating cells with 1 μM DCFDA, then staining cells for MDSCs and analyzing MDSCs for ROS fluorescence by flow cytometry. Gating was set based on NOHA-treated MDSCs. (B) Splenic MDSCs were cultured as in panel A, with the addition of an aIEP or a control IgY, with mean fluorescence intensities (MFIs) indicated. (C) Blood and spleens were harvested from BALB/c^nu/nu mice harboring either DA-3/TM or DA-3/sec tumors, and cells were directly treated with DCFDA without in vitro culture, and stained and gated for MDSC analysis of ROS production. Histograms were overlayed and MFIs are indicated. (D) Blood MDSCs from DA-3/sec or DA-3/TM tumor-bearing BALB/c^nu/nu mice were fixed on slides and stained for histology. (E) Splenocytes from DA-3/sec or DA-3/TM tumor-bearing BALB/c^nu/nu mice were stained and gated for MDSCs, and the indicated markers were analyzed, with MFIs listed. Results are representative of at least 3 experiments.