Figure 5.

MyD88 adaptive molecule plays a role in tumor exosome-mediated promotion of B16 tumor cell growth in the lung and in tumor chemotaxis. Seven-week-old wild-type B6 or MyD88 knockout mice were injected intravenously with B16 exosomes (100 μg/mouse in 100 μl of PBS) or E-control twice a week for three weeks. One day after the last injection, the mice were injected intravenously with B16–luc cells (1 × 10⁵). Twenty-one days after B16-luc injection, the mice were imaged at 0 and 4 hours (A) after injection of D-luciferin, and the total photon count per minute (photons per minute) was calculated (nine animals/group, three representative mice are shown) by using Living Image software (A; left). The growth potential of injected B16–luc cells was determined by dividing photon emissions at 4 hours by the photon emissions at 0 hours (A; right) for mice treated with exosomes or the E-control. To determine the specificity of B16 exosome-mediated promotion of B16-luc growth in the lung, mice were treated with EF-exosomes or B16 exosomes by using an identical protocol as described in A. In vivo luciferase activity was measured as described in A. All results in B are based on two independent experiments with data pooled for mice in each experiment. Results in both A and B are presented as the mean ± SEM; **P < 0.01. Seven-day cultured BM precursor cells isolated from B6 wild-type mice or BALB/c mice and treated as described in Figure 2A were co-cultured with B16-luc or 4T-1-luc in a 24-well transwell plate. Twenty-four hours later the cells in the bottom chamber were harvested, lysed, and luciferase activity was determined. Results are presented as percent of luciferase activity. Data presented are mean ± SEM of triplicates wells for three independent experiments (C). Supernatants of seven-day cultures treated as described in Figure 1, A–C, were collected, and the amount of CCL2 (D) was measured by using a standard ELISA. Data represent mean ± SEM of three samples in three independent experiments. **P < 0.01. Seven-day cultured BM precursor cells treated as described in Figure 2A were co-cultured with B16-luc in the presence of either anti-mouse CCL2 (1.0 μg/ml) or a control hamster IgG in a 24-well transwell plate. Twenty-four hours later the cells in the bottom chamber were harvested, lysed, and luciferase activity was determined. Results are presented as percent of luciferase activity. Data presented are mean ± SEM of triplicates wells for three independent experiments (E).