Fig. 3. Nr4a1-expressing patrolling monocytes reduce tumor metastasis and engulf tumor material in the lung.

(A) In vivo imaging (Left), and quantification (Right) of tumor in lungs of CSF1R-Cre⁻Nr4a1^fl/fl (CSF1R-Cre⁻) or CSF1R-Cre⁺Nr4a1^fl/fl (CSF1R-Cre⁺) mice 7 days after IV injection of 3×10⁵ B16F10-luciferase tumor cells (n=6 mice per group, *=p<0.01; experiment replicated 2 times). (B) Quantification of the number of tumor metastases per lungs of CSF1R-Cre⁻Nr4a1^fl/fl (CSF1R-Cre⁻) and CSF1R-Cre⁺Nr4a1^fl/fl (CSF1R-Cre⁺) mice 7 days after IV injection of 3×10⁵ B16F10-YFP tumor cells (n=8 mice per group, *=p<0.01). (C-D) Nr4a1^−/− mice were injected IV with 5×10⁵ wild-type Ly6C⁻ PMo, Ly6C⁺ inflammatory monocytes, or PBS at day 0. On day 1, 3×10⁵ B16F10-luciferase tumor cells were injected IV and tumor metastasis and growth were measured by in vivo imaging at day 8. Representative in vivo imaging (C), and quantification (D) of B16F10-luciferase metastasis 8 days after monocyte transfer and 7 days after tumor transfer in wild-type (WT) or Nr4a1^−/− mice. (Combined data from 5 separate experiments with n=2 mice per group; *=p<0.01 statistically different than WT; **=p<0.05 statistically different than Nr4a1^−/−). (E) Imaging of tumor material uptake in lung by Nr4a1-GFP^high monocytes 24 hrs after IV injection of LLC-RFP tumor cells. Representative higher magnification images to right. (F) Uptake of LLC-RFP tumor material by CX3CR1-GFP^highLy6C⁻ PMo after 24 hrs of co-culture. Note that Nr4a1-GFP expression is primarily nuclear so monocyte cell membranes are not visible in images (G) Representative flow plot (Left) and quantification (Right) of tumor material uptake by all monocytes in the lung 24 hrs after IV tumor injection of 3×10⁵ LLC-RFP cells (n=4 mice per goup; *=p<0.01; experiment replicated 3 times).