CXCR3+ monocytes/macrophages are required for establishment of pulmonary metastases

Abstract

We present a new foundational role for CXCR3⁺ monocytes/macrophages in the process of tumor engraftment in the lung. CXCR3 is associated with monocytic and lymphocytic infiltration of inflamed or tumor-bearing lung. Although the requirement for tumor-expressed CXCR3 in metastatic engraftment has been demonstrated, the role of monocyte-expressed CXCR3 had not been appreciated. In a murine model of metastatic-like melanoma, engraftment was coordinate with CXCR3⁺ monocyte/macrophage accumulation in the lungs and was sensitive to pharmacologic inhibition of CXCR3 signaling. Tumor engraftment to lung was impaired in CXCR3^-/- mice, and transient reconstitution with circulating CXCR3-replete monocytes was sufficient to restore engraftment. These data illustrate the paradoxical pro-tumor role for CXCR3 in lung immunobiology wherein the CXCR3 axis drives both the anti-tumor effector cell chemoattraction and pro-tumor infiltration of the lungs and suggests a potential therapeutic target for lung-tropic metastasizing cancers.

Figure 1. CXCR3 enables the establishment of metastatic-like melanoma in lungs.

Mice were intravenously injected with 3 × 10⁵ B16F10 cells. (A) Expression of the melanocyte/melanoma-specific gene product tyrosinase in lungs in WT and CXCR3^−/− mice, assessed 12d post-tumor challenge. (B) The number of visible lung metastases in WT and CXCR3^−/− mice, assessed 12d post-tumor challenge. (C) Expression of tyrosinase in lungs of WT and CXCR3^−/− mice, with or without tumor, assessed at 24 h post tumor-challenge. In gene expression experiments, qPCR data were normalized to GAPDH expression. Data are representative of two independent experiments with similar outcomes, with n = 7–10 mice/group in each study (Panels A and B) and n = 4–7 mice/group (Panel C). Differences were assessed by T-test as indicated: *p < 0.05; **p < 0.01; ****p < 0.0001.

Figure 2. Removal of circulating monocytes ablates melanoma engraftment in the lung.

Mice were intravenously injected with 3 × 10⁵ B16F10 cells. Some mice received a macrophage-depleting treatment (chlodronate-containing liposomes) prior to tumor injections. Engraftment was measured by relative expression of tyrosinase, normalized to GAPDH, 24 h post-injection of tumor. Data are representative of two independent experiments with similar outcomes, with n = 4–7 mice/group in each study. Differences were assessed by T-test as indicated: ****p < 0.0001.

Figure 3. Tumor-induced accumulation of monocytes in lung is compromised in CXCR3^−/− mice.

Mice were intravenously injected with 3 × 10⁵ B16F10 cells. Twenty-four hours following B16F10 tumor injection, single cell suspensions of lungs were analyzed by flow cytometry. (A) Representative staining for circulatory-type monocytes (CD45⁺CD11b⁺Ly6C⁺) in lungs. Gated on viable CD45⁺ events. (B) Total numbers of lung monocytes. (C) Proportion of lung monocytes. Differences were assessed by T-test as indicated ***p < 0.001.

Figure 4. Tumor-induced accumulation of macrophages in lung is compromised in CXCR3^−/− mice.

Mice were intravenously injected with 3 × 10⁵ B16F10 cells. Twenty-four hours following B16F10 tumor injection, single cell suspensions of lungs were analyzed by flow cytometry. (A) Representative staining for macrophages (CD45⁺CD11b⁺ F4/80⁺) in lungs. Gated on viable CD45⁺ events. (B) Total numbers of lung macrophages. (C) Proportion of lung macrophages. Differences were assessed by T-test as indicated ***p < 0.001; ****p < 0.0001.

Figure 5. CXCR3 signaling blockade with AMG487 inhibits the tumor-associated accumulation of macrophages or monocytes in lung.

AMG487 or vehicle (n = 8 per group) was injected subcutaneously, −1 d and 0 d before B16F10 tumor injection. Mice were intravenously injected with 3 × 10⁵ B16F10 cells or HBSS control. Twenty-four hours later, lungs and spleen were harvested for flow cytometry. F4/80⁺CD11b⁺ cells were assessed in the lung (A) and spleen (B) and circulating CD11b⁺Ly6C⁺ cells were assessed in the lung (C) and spleen (D). In all panels, differences were assessed by T-test as indicated: ***p < 0.001.

Figure 6. CXCR3-expressing monocytes are required for tumor engraftment in lungs.

(A) Mice were intravenously injected with 3 × 10⁵ B16F10 cells, then selectively depleted of monocytes/macrophages and reconstituted with WT or CXCR3^−/− monocytes. (B) Tyrosinase expression, measured in the lung 24 h after tumor inoculation. Gene expression data were normalized to GAPDH. Data are representative of two independent experiments with similar outcomes, with n = 4–8 mice/group in each study. Differences were assessed by T-test as indicated: *p < 0.05; **p < 0.01.