COX-2 and prostaglandin EP3/EP4 signaling regulate the tumor stromal proangiogenic microenvironment via CXCL12-CXCR4 chemokine systems

Abstract

Bone marrow (BM)-derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)-2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3-/- mice and EP4-/- mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4+ fibroblasts, CXCL12+, and/or CXCR4+ stromal cells as well. Immunofluorescent analyses revealed that CXCL12+CXCR4+S100A4+ fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE2-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under the control of prostaglandins.

Figure 1

COX-2–dependent stromal formation and angiogenesis in the tumor implantation model, and expression of chemokines and chemokine receptors in tumor stroma. A: Time course of tumor growth in the absence (−; unfilled) and presence (+; filled) of Celecoxib. The day of inoculation was defined as day 0. Celecoxib significantly attenuated tumor growth on days 7, 14, and 21. n = 5, 10, and 7 per group on days 7, 14, and 21, respectively. B: Typical appearance of tumors excised on day 14. Scale bars = 5.0 mm. C and D: Angiogenesis (MVD and MVA) on day 14. E: Stromal formation (stromal thickness) on day 14. F and G: The mRNA expression of CXCL12 and CXCR4 in tumor stroma on day 14. H: CXCL12 protein expression in tumor stroma on day 14. n = 10 per group in C–H. The results represent means ± SEM. *P < 0.05; **P < 0.01 by Student t test (compared with vehicle-treated mice on the same day).

Figure 2

COX-2–dependent enhancement of stromal formation and angiogenesis around micropore chambers, and the involvement of CXCL12/CXCR4 signaling. A: Typical appearance of granulation tissues formed around micropore chambers. Diameter of chamber ring, 13 mm. B and C: Angiogenesis formed around micropore chambers, as measured by MVD and MVA. D and E: Stromal wet weight and stromal thickness. F, G and I: mRNA expression of CXCL12, CXCR4, and VEGF-A, respectively. H: CXCL12 protein level in granulation tissues formed around micropore chambers. J: Immunofluorescence analysis of CXCL12 (left) and S100A4 (middle) expression in tumor stoma from vehicle-treated mice (top row) and Celecoxib-treated mice (bottom row). Almost all (99.7%) of CXCL12⁺ cells are positive for S100A4; arrows; merged signal, yellow (right). Arrowheads indicate single labeled cells with S100A4. Scale bars = 20 μm. Quantification of each labeled cells is shown. Treatment with Celecoxib decreased the number of CXCL12⁺S100A4⁺ cells. K: mRNA expression of S100A4, F4/80, and CD3ε in granulation tissues. The results represent means ± SEM from 10 mice per group. *P < 0.05; **P < 0.01 by Student t test (compared with vehicle-treated mice).

Figure 3

A–R: Double labeling analysis of CXCL12 or CXCR4 with S100A4, F4/80, or CD3ε in tumor stoma 14 days after tumor implantation. Among the CXCL12-positive cells in the tumor stroma, 94.4% are S100A4-positive (A–C), 6.4% are F4/80-positive (G–I), and 0% are CD3ε-positive (M–O). Among CXCR4-positive cells, 87.8% are S100A4-positive (D–F), 5.8% are F4/80-positive (J–L), and 0% are CD3ε-positive (P–R). Most CXCL12⁺ or CXCR4⁺ stromal cells were S100A4-positive fibroblasts. S–U: Double labeling assay of GFP and S100A4 in tumor stoma of tumor implantation model in GFP-BM chimeric mice. 92.3% of GFP⁺ cells were positive for S100A4. And GFP⁺S100A4⁺ cells occupied 89.6% of S100A4-positive stromal fibroblasts. Scale bars = 20 μm.

Figure 4

CXCL12 (SDF-1α) enhances granulation tissue formation that mimics the stromal reaction and angiogenesis in a Matrigel implantation model. A: Typical appearance of stromal formation and angiogenesis formed around a Matrigel. Scale bars = 10 mm. B and C: Angiogenesis (MVD and MVA) was significantly promoted by CXCL12 application. D: Stromal thickness was markedly increased by CXCL12 application. E: mRNA expression of VEGF-A in stroma formed around a Matrigel. The results represent means ± SEM from 11 mice per group. *P < 0.05; **P < 0.01 by Student t test (compared with vehicle-treated mice).

Figure 5

CXCR4-neutralizing antibody (2B11) reduces angiogenesis and granulation tissue formation around micropore chambers. A: Typical appearance of stromal formation and angiogenesis formed around micropore chambers. Diameter of chamber ring, 13 mm. B and C: Angiogenesis (MVD and MVA) in the granulation tissues around micropore chambers. D and E: Stromal formation (stromal wet weight and stromal thickness). F: mRNA expression of VEGF-A in the granulation tissues. G, H and I: mRNA expression of S100A4 (G), F4/80 (H), and CD3ε (I) in granulation tissues. n = 11 in IgG-treated group and n = 15 in 2B11-treated group. The results represent means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by Student t test (compared with control IgG-treated mice).

Figure 6

Identification of EP receptor signaling pathways that facilitate of stromal formation in the tumor implantation model. A and B: Stromal wet tissue weight and stromal thickness in each EP receptor knockout mice. C and D: mRNA expression of CXCL12 and CXCR4 in stromal tissue in each EP receptor knockout mice. E: CXCL12 protein level in stromal tissue of wild-type, EP3^−/−, and EP4^−/− mice. F: mRNA expression of S100A4 in stromal tissue in EP receptor knockout mice. n = 10, 4, 5, 8, 8, and 7 in wild-type (WT), EP1^−/−, EP2^−/−, EP3^−/−, EP4WT, and EP4^−/−, respectively. The results represent means ± SEM. *P < 0.05; **P < 0.01 by Student t test (compared with wild-type mice in EP1, 2, and 3^−/− mice, EP4WT mice were used for comparison as to EP4^−/− mice).

Figure 7

Effects of EP1, EP2, EP3, and EP4 specific agonists on CXCL12 gene expression by L929 fibroblasts. L929 fibroblasts (3 × 10⁵ cells per well) cultured in six-well plates were incubated for 24 hours with the respective agonists, then CXCL12 gene expression was measured. Data were expressed as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 by Student t test (compared with vehicle-treated cells).