TLR2-mediated expansion of MDSCs is dependent on the source of tumor exosomes

Abstract

Exosomes released from tumor cells having been shown to induce interleukin-6 release from myeloid-derived suppressor cells in a Toll-like receptor 2/Stat3-dependent manner. In this study, we show that exosomes released from tumor cells re-isolated from syngeneic mice are capable of inducing interleukin-6 in a Toll-like receptor 2-independent manner, whereas the data generated from exosomes of tumor cells having undergone numerous in vitro passages induce interleukin-6 in a Toll-like receptor 2-dependent manner. This discrepancy may be due to the source of tumor cells used to generate the exosomes for this study. These results suggest that exosomes released from tumor cells that are not within a tumor microenvironment may not realistically represent the role of tumor exosomes in vivo. This is an important consideration since frequently passing tumor cells in vivo is an accepted practice for studying tumor exosome-mediated inflammatory responses.

Figure 1

TLR2-mediated tumor exosome blocking of the differentiation of GM-CSF-stimulated BM precursor cells is dependent on the source of tumor exosomes. Erythrocyte-depleted BM cells of WT B6 mice or B6 mice with MyD88 or TLR2 knockout were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 20 ng/ml recombinant mouse GM-CSF. B16 exosomes or bovine serum albumin (10 μg/ml) was added at day 0 after the addition of the GM-CSF to the cell cultures. After 7 days in culture, the cells were analyzed by fluorescence-activated cell sorting for the expression of CD11b and Gr-1. One representative of five independent experiments is shown (A, left panel). All data are given as the average values ± SEM obtained for five samples in three independent experiments (A, right panel). B: Supernatants of 7-day cultures treated as described in A were collected, and the amount of IL-6 was measured using a standard ELISA. Data represent the mean ± SEM of five samples in three independent experiments, **P < 0.01. To determine the effect of B16 exosome-mediated induction on phosphorylated Stat3, erythrocyte-depleted BM cells of WT B6 mice or B6 mice with a MyD88 or TLR2 knockout were treated with B16 exosomes or bovine serum albumin (10 μg/ml) in the presence of 20 ng/ml recombinant mouse GM-CSF. After 48 hours in culture, phosphorylated Stat3 and total Stat3 were detected in cells by Western blot analysis (C, left panel). The signal intensity of each protein analyzed was quantified using an Odyssey infrared imaging system (LI-COR). The ratios of signal intensity of phosphorylated Stat3 (after normalized with total Stat3) in cells treated with either C-exo or P-exo against bovine serum albumin were calculated and plotted (C, right panel). The data are presented as the mean of three independent experiments.

Figure 2

TLR2-dependent exosome factor is heat stable and chloroform soluble. Erythrocyte-depleted BM cells of WT B6 mice were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 20 ng/ml recombinant mouse GM-CSF. Lipids extracted from 10 μg B16 exosomes, 100°C heat-inactivated B16 exosomes (10 μg), or 10 μg B16 exosomes without treatment were added at day 0 after the addition of GM-CSF to the cell cultures. The supernatants of 7-day cultures were collected, and the amount of IL-6 was measured using a standard ELISA (A). B16 exosomes purified from B16 tumor cells (passage number >40) pre-co-cultured with CD3-activated spleen T cells (CT-exo), C-exo, or bovine serum albumin (10 μg/ml) were added at day 0 after the addition of GM-CSF to the cell cultures. Supernatants of 7-day cultures were collected, and the amount of IL-6 was measured using a standard ELISA (B). Data represent the mean ± SEM of five samples in three independent experiments, **P < 0.01.