Tumor angiogenesis mediated by myeloid cells is negatively regulated by CEACAM1

Abstract

Bv8 (prokineticin 2) expressed by Gr1(+)CD11b(+) myeloid cells is critical for VEGF-independent tumor angiogenesis. Although granulocyte colony-stimulating factor (G-CSF) has been shown to be a key inducer of Bv8 expression, the basis for Bv8 production in driving tumor angiogenesis is undefined. Because the cell adhesion molecule CEACAM1, which is highly expressed on Gr1(+)CD11b(+) myeloid cells, is known to regulate G-CSF receptor (G-CSFR) signaling, we hypothesized that CEACAM1 would regulate Bv8 production in these cells. In support of this hypothesis, we found that Bv8 expression was elevated in Gr1(+)CD11b(+) cells from Ceacam1-deficient mice implanted with B16 melanoma, increasing the infiltration of Gr1(+)CD11b(+) myeloid cells in melanoma tumors and enhancing their growth and angiogenesis. Furthermore, treatment with anti-Gr1 or anti-Bv8 or anti-G-CSF monoclonal antibody reduced myeloid cell infiltration, tumor growth, and angiogenesis to levels observed in tumor-bearing wild-type (WT) mice. Reconstitution of CEACAM1-deficient mice with WT bone marrow cells restored tumor infiltration of Gr1(+)CD11b(+) cells along with tumor growth and angiogenesis to WT levels. Treatment of tumor-bearing WT mice with anti-CEACAM1 antibody limited tumor outgrowth and angiogenesis, albeit to a lesser extent. Tumor growth in Ceacam1-deficient mice was not affected significantly in Rag(-/-) background, indicating that CEACAM1 expression in T and B lymphocytes had a negligible role in this pathway. Together, our findings show that CEACAM1 negatively regulates Gr1(+)CD11b(+) myeloid cell-dependent tumor angiogenesis by inhibiting the G-CSF-Bv8 signaling pathway.

Figure 1. Tumor growth and angiogenesis are enhanced CEACAM1^−/− mice

(A) B16 tumor cells were injected subcutaneously into C57BL/6 or Ceacam1^−/− mice. Data represent mean ± SEM. **0.001<P ≤ 0.01 (n=8 mice per group from three independent experiments). (B) Blood vessels in tumors were counted based on immunohistological analysis with anti-CD31 of frozen tumor tissue collected from mice in (A) after 17 days. More than 8 fields of view were analyzed. Data represent mean ± SEM. ***P ≤ 0.001. (C) Immunohistochemistry staining of mouse B16 tumor tissue collected from mice in (A) after 17 days with anti-CD31 antibody. Bar = 200µm. (D) Representative photos of Matrigel plugs isolated from Ceacam1^−/− (left) or C57BL/6 (right) mice after 6 days implantation. (E) Quantitation of hemoglobin in Matrigel plugs containing B16 cells from Ceacam1^−/− mice or C57B/6 controls. (F) Quantitation of vascular surface area in Matrigel plugs containing B16 cells from Ceacam1^−/− mice or C57B/6 controls. Data represent mean ± SEM. ***P ≤ 0.001 (n=6 mice per group from three independent experiments).

Figure 2. Enhanced tumor growth and angiogenesis is dependent on bone marrow-derived cells but independent of T and B cells

(A) Recipient Ceacam1^−/− and C57BL/6 mice were lethally irradiated and reconstituted with bone marrow from donor C57BL/6 or Ceacam1^−/− mice. After 6 weeks, chimeras were injected s.c. with B16F10 melanoma cells and tumor growth in chimeras measured. Data represent mean ± SEM. *0.01< P ≤0.05, **0.001< P ≤ 0.01 (n=8 mice per group from two independent experiments). (B) Blood vessels in tumors were counted on frozen tissue after 17days. More than 8 fields of view were analyzed. Data represent mean ± SEM. ***P ≤ 0.001. (C) Immunohistochemisty staining of mouse B16 tumor tissue from chimeras with anti-CD31 antibody. Bar = 200µm. (D) B16 tumors were injected s.c. into Ceacam1^−/−Rag1^−/− and Rag1^−/− and tumor growth was measured for 17 days. Data represent mean ± SEM. *0.01< P ≤0.05, **0.001<P ≤ 0.01 (n=10 mice per group from two independent experiments). (E) Blood vessels in tumors were counted on frozen tumor tissue collected from mice in (D) after 17days. Data represent mean ± SEM. ***P ≤ 0.001. (F) Immunohistochemisty staining of mouse B16 tumor tissue collected from mice in (A) after 17 days with anti-CD31. Bar = 200µm.

Figure 3. Infiltration of Gr1⁺CD11b⁺ myeloid cells is enhanced in tumors of Ceacam1^−/− mice

(A) Immunofluorescent staining with Gr1⁺ antibody (Red) plus DAPI (blue) of frozen tumor tissue harvested after 17 days. Bar = 200µm. (B) Quantitation of Gr1⁺ cells in tumor was taken from immunofluoresecent staining in (A). More than 10 fields of view were analyzed. Data represent mean ± SEM. ***P ≤ 0.001. (C) Single-cell suspensions were prepared from B16 tumors harvested 17 days after tumor implantation. The mean percentage of Gr1⁺CD11b⁺ cells in tumor, blood, spleen and bone marrow from Ceacam1^−/− and C57BL/6 tumor-bearing mice is shown in bar graphs. Data represent mean ± SEM, *0.01< P ≤0.05, **0.001<P ≤ 0.01, ***P ≤ 0.0001 (n=6 mice per group from three independent experiments). (D) Representative flow cytometric analysis showing Gr1⁺CD11b⁺ myeloid population in chimeras tumor-bearing mice (left) and in Ceacam1^−/−Rag1^−/− or Rag1^−/− tumor bearing mice (right), (n=6 mice per group from three independent experiments). (E) Immunofluorescent staining with Gr1⁺ antibody (Red) plus DAPI (blue) of frozen Matrigel plugs harvested after 6 days in CEACAM1−/− mice vs C57B/6 controls. Quantitation is shown to the right (**P<0.01). Bar = 200µm.

Figure 4. Gr1⁺CD11b⁺ myeloid cells are sufficient and necessary for enhanced tumor growth and angiogenesis in Ceacam1^−/− mice

(A) Wild type mice injected s.c. with B16 tumor cells mixed with Gr1⁺CD11b⁺ cells enriched from tumor-bearing Ceacam1^−/− mice (Ceacam1^−/− +B16) or WT mice (C57BL/6+B16) or B16 alone (B16 control). Data represent mean ± SEM, *0.01< P ≤0.05, *** P ≤ 0.001 (n=6 mice per group from three independent experiments). (B) Blood vessels in tumors were counted from mice in (A) after 17 days. More than 8 fields of view were analyzed. Data represent mean ± SEM. **0.001< P ≤ 0.01, ***P ≤ 0.001. (C) Immunohistochemistry staining (anti-CD31 antibody) of mouse B16 tumor tissue collected from mice in (A) after 17 days. (D) Gr1⁺CD11b⁺ cells were depleted by anti-Gr1 Ab, and compared to untreated animals. Data represent mean ± SEM. *0.01< P ≤0.05, **0.001< P ≤ 0.01, NS (no significance) (n=8 mice per group from two independent experiments). (E) Frozen tumor tissue harvested after 17 days were stained with anti-CD31 antibody. Data represent mean ± SEM. ***P ≤ 0.001. (F) Immunohistochemistry staining (anti-CD31) of mouse B16 tumor tissue collected from mice in (D) after 17 days of treatment with anti-CD31 antibody. Bar = 200µm.

Figure 5. Bv8 is elevated in Ceacam1^−/− Gr1⁺CD11b⁺ myeloid cells

(A) G-CSF and VEGF secretion from B16 tumors from Ceacam1^−/− and C57BL/6 was assessed by ELISA after 12-h culture. Data represent mean ± SEM *0.01< P ≤0.05, (B) Real-time PCR analysis of Bv8 mRNA transcript levels in Gr1⁺CD11b⁺ cells enriched from spleen and tumor of Ceacam1^−/− and C57BL/6 tumor-bearing mice. Data represent mean ± SEM *0.01< P ≤0.05, **0.001< P ≤ 0.01. (C) Real-time PCR analysis of VEGF mRNA transcript levels in Gr1⁺CD11b⁺ cells enriched from tumor and spleen of Ceacam1^−/− and C57BL/6 tumor-bearing mice. Data represent mean ± SEM. (D) Bv8 mRNA levels are upregulated by G-CSF in Ceacam1^−/− Gr1⁺CD11b⁺ cells. Real-time PCR showing Bv8 mRNA transcript levels in Gr1⁺CD11b⁺ or Gr1⁻CD11b⁻ cells enriched from Ceacam1^−/− or C57BL/6 bone marrow, with or without G-CSF treatment (50 ng/ml). Data represent mean ± SEM. (E) Immunoblot analysis of Bv8 in Ceacam1^−/− and C57BL/6 Gr1⁺CD11b⁺ cells with or without G-CSF treatment (50 ng/ml). Qunatitation by densitomtery and normalized to GAPDH.

Figure 6. Tumor growth and angiogenesis are reduced in anti-Bv8 treated Ceacam1^−/− mice

(A) Anti-Bv8 blocking antibody reduced tumor growth (n=6 mice per group) in Ceacam1^−/− mice bearing B16 tumors. Data represent mean ± SEM. *0.01< P ≤0.05, **0.001< P ≤ 0.01. (B) Blood vessels in tumors were counted based on immunohistological analysis with anti-CD31 of frozen tumor tissue collected from mice in (A) after 17 days. More than 8 fields of view were analyzed. Data represent mean ± SEM. **0.001< P ≤ 0.01. (C) Immunohistochemistry staining of mouse tumor tissue collected from mice in (A) after 17 days with anti-CD31 antibody. Bar = 200µm. (D) Immunofluorescent staining of tumor associated Gr1⁺ cells. Bar = 200µm.

Figure 7. Tumor growth and angiogenesis are reduced in ant-VEGF or combined anti-VEGF anti-Bv8 treated Ceacam1^−/− mice

(A) Anti-VEGF, anti-Bv8, or combined blocking antibodies reduced tumor growth (n=6 mice per group) in Ceacam1^−/− mice bearing B16 tumors. Data represent mean ± SEM, *P ≤0.01. (B) Blood vessels in tumors were counted based on immunohistological analysis with anti-CD31 of frozen tumor tissue collected from mice in (A) after 17 days. More than 8 fields of view were analyzed. Data represent mean ± SEM. **0.001< P ≤ 0.01. (C) Immunofluorescent staining of tumor associated Gr1⁺ cells. Data represent mean ± SEM. **0.001< P ≤ 0.01.