Patrolling monocytes control tumor metastasis to the lung

Abstract

The immune system plays an important role in regulating tumor growth and metastasis. Classical monocytes promote tumorigenesis and cancer metastasis, but how nonclassical "patrolling" monocytes (PMo) interact with tumors is unknown. Here we show that PMo are enriched in the microvasculature of the lung and reduce tumor metastasis to lung in multiple mouse metastatic tumor models. Nr4a1-deficient mice, which specifically lack PMo, showed increased lung metastasis in vivo. Transfer of Nr4a1-proficient PMo into Nr4a1-deficient mice prevented tumor invasion in the lung. PMo established early interactions with metastasizing tumor cells, scavenged tumor material from the lung vasculature, and promoted natural killer cell recruitment and activation. Thus, PMo contribute to cancer immunosurveillance and may be targets for cancer immunotherapy.

Fig. 1. Nr4a1-GFP^high monocytes patrol the vasculature, and interact with tumor in the lung

(A) Quantification of Nr4a1-GFP^high PMo per μl of blood volume in lung (Untreated), 4 hrs or 24 hrs after IV LLC-RFP transfer (n=5 mice per group). (B) Quantification of Nr4a1-GFP^high monocyte movement in lung before (Untreated), 4 hrs or 24 hrs after LLC-RFP tumor injection. Monocyte tracks transposed to a common origin from a representative 20 min movie (left, scale bar=100 μm), and quantification of median speed of monocytes (right, combined speed data from analysis of 3 separate mice; *=p<0.001 lower than untreated; **=p<0.001 than 4hr tumor). (C) Representative gating of Nr4a1-GFP^highCD11b⁺ cells from all Live CD45⁺CD11c^low cells 24 hrs after IV LLC-RFP transfer. (D) Representative confocal image of Nr4a1-GFP^high monocytes (Green) interacting with LLC-RFP cells (Red) in the lung 7 days after IV LLC-RFP transfer. Immune cells in the vasculature were labeled with IV injected anti-CD45 antibody (Blue). (E) Quantification of free (>100 μm from tumor site) and tumor-associated (<50 μm from tumor site) Nr4a1-GFP^high monocytes in the lung at various time points after tumor injection (combined analysis of 5 mice per group; p<0.01 for each tumor-associated area compared to free tumor area for each time point).

Fig. 2. Increased lung metastasis of tumors in Nr4a1^−/− mice

(A) In vivo luciferase detection in wild-type control (WT) and Nr4a1^−/− mice 24 hrs after IV injection of 5×10⁵ B16F10 melanoma cells expressing luciferase (Left) and quantification (Right) (*=p<0.03, representative experiment with 5 mice per group). (B) In vivo luciferase detection (Left), and quantification (Right), in WT and Nr4a1^−/− mice 7 days after IV injection with 3×10⁵ B16F10-luciferase cells (*=p<0.001, n=18 mice per group combined from 3 separate experiments). (C) Number of spontaneous tumor metastases per 5000 μm² of lung surface 28 days after SubQ injection of 1×10⁵ B16F10-YFP cells (*=p<0.01, n=7 mice per group). (D-E) Lung tumor metastasis in MMTV-PyMT mice reconstituted with WT (WT:PyMT) or Nr4a1^−/− (Nr4a1^−/− :PyMT) bone marrow. (D) Representative MMTV-PyMT mouse lung histology stained with hematoxylin and eosin (Left), and quantification of number of spontaneous lung metastases per 5000 μm² of lung surface (Right). *=p<0.05 n=12 for WT and n=15 for Nr4a1^−/−. (E) Quantification of primary breast tumor growth in MMTV-PyMT mice. N=12 for WT and n=15 for Nr4a1^−/−.

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).

Fig. 4. Patrolling monocytes detect tumor material in a CX3CR1-dependent manner and recruit NK cells to the lung tumor environment

(A) Ratio of fluorescent intensity of tumor material engulfed by PMo (black) to fluorescent intensity of whole tumor (black and grey) 3 hrs after IV LLC tumor injection. LLC tumors were labeled with either CellTrace Violet control dye (Control) or a pH-sensitive pHrodo Red dye (pHrodo) and then IV injected in a 1:1 ratio into a wild-type mouse (n=3 mice per group of representative experiment replicated 3 times). Representative tracking (B) and median speed (C) of Cx3cr1^−/− or Cx3cr1^−/+ monocyte movement 24 hrs after IV tumor injection in the lung. Monocyte tracks transposed to a common origin from representative 20 min movies (scale bar=100 μm, representative tracks shown from one mouse, median speed calculated from tumor areas analyzed in 3 separate mice per group, *=p<0.001). (D) Percentage of Ly6C⁻ PMo containing LLC-RFP tumor material in the lung 3 hrs after IV injection of tumor into representative wild-type (WT), Cx3cr1^−/−, or Tlr7^−/− mice (left), and quantification of tumor material uptake (right) (n=3 per group; *p<0.001 versus WT). (E) Percentage of CD31⁺ CX3CL1⁺ lung endothelial cells isolated from Untreated (UN) or 24 hrs or 7 days after IV injection of B16F10-YFP tumor cells into CX3CL1-mCherry mice. (F) Representative imaging of CX3CL1-mCherry (Red) expression in lung 24 hrs after IV injection of B16F10-YFP tumor cells (Green) in CX3CL1-mCherry mice. CD45⁺ immune cells are labeled in blue. (G) Relative chemokine mRNA expression in Ly6C⁺ or Ly6C⁻ monocytes isolated from lung by FACS 24 hrs after IV B16F10 tumor injection (monocyte populations isolated from three separate mice; *=p<0.01; experiment repeated 3 times). (H) Percentage of natural killer (NK) cells in the 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).