Noninvasive imaging reveals inhibition of ovarian cancer by targeting CXCL12-CXCR4

Abstract

Patients with metastatic ovarian cancer continue to have a dismal prognosis, emphasizing the need for new strategies to identify and develop new molecular targets for therapy. Chemokine CXCL12 and its receptor CXCR4 are upregulated in metastatic ovarian cancer cells and the intraperitoneal tumor microenvironment. CXCL12-CXCR4 signaling promotes multiple steps in proliferation and dissemination of ovarian cancer cells, suggesting that targeted inhibition of this pathway will limit tumor progression. To investigate CXCL12-CXCR4 signaling in ovarian cancer and establish effects of inhibiting this pathway on tumor progression and survival, we designed a Gaussia luciferase complementation imaging reporter system to detect CXCL12 binding to CXCR4 in ovarian cancer cells. In cell-based assays, we established that the complementation imaging reporter could detect CXCL12 binding to CXCR4 and quantify specific inhibition of ligand-receptor interaction. We monitored CXCL12-CXCR4 binding and inhibition in a mouse xenograft model of metastatic human ovarian cancer by imaging Gaussia luciferase complementation and assessed tumor progression with firefly luciferase. Bioluminescence imaging studies in living mice showed that treatment with AMD3100, a clinically approved inhibitor of CXCL12-CXCR4, blocked ligand-receptor binding and reduced growth of ovarian cancer cells. Treatment with AMD3100 also modestly improved overall survival of mice with metastatic ovarian cancer. The Gaussia luciferase complementation imaging reporter system will facilitate further preclinical development and optimization of CXCL12-CXCR4 targeted compounds for treatment of ovarian cancer. Our research supports clinical translation of existing CXCR4 inhibitors for molecular therapy for ovarian cancer.

Figure 1

HeyA8 ovarian cancer cells express functional CXCR4. (A) Cell surface levels of CXCR4 on HeyA8 cells were determined by flow cytometry. Dark line indicates unstained; light line, isotype antibody control; and intermediate line, CXCR4 antibody. (B) HeyA8 cells were serum starved overnight and then incubated with 2 or 100 ng/ml CXCL12 for 10 minutes. Cell lysates were probed for phosphorylated AKT (p-AKT, active form) and total AKT as a control for protein loading.

Figure 2

Gaussia luciferase complementation detects CXCL12-CXCR4 binding in cell-based assays. (A) Schematic diagram of complementation system. The C-terminus of CXCL12 is fused to the C-terminal fragment of Gaussia luciferase (CXCL12-CG), and the N-terminal fragment of Gaussia is fused to the extracellular N-terminus of CXCR4 (NG-CXCR4). These orientations of fusion proteins position NG and CG fragments in the extracellular space. CXCL12 binding to CXCR4 brings together NG and CG enzyme fragments, reconstituting an active enzyme to produce light. (B) Flow cytometry shows expression of CXCR4 on the surface of NG-CXCR4 cells. Dark line indicates unstained; light line, isotype antibody control; and intermediate line, CXCR4 antibody. (C) HeyA8-NG-CXCR4 cells were cocultured with equal numbers of HeyA8-CXCL12-CG (CXCL12-CG/NG-CXCR4) or control cells secreting unfused CG (CG/NG-CXCR4) for 2 hours before quantifying Gaussia luciferase bioluminescence. Photon flux was normalized to relative amounts of total protein per well, and data were expressed as mean values ± SEM for each coculture (n = 4 per condition). (D) Cocultures of HeyA8-CXCL12-CG/NG-CXCR4 or HeyA8-CG/NG-CXCR4 cells were incubated for various periods through 2 hours before quantifying Gaussia luciferase bioluminescence as described in C (n = 4 per condition). **P < .01. ***P < .005.

Figure 3

AMD3100 inhibits bioluminescence from CXCL12-CXCR4 binding. (A) Cocultures of HeyA8 cells (CXCL12-CG/NG-CXCR4 or CG/NG-CXCR4) were incubated for increasing periods with 1 µM AMD3100, a CXCR4 inhibitor, or CCX771, an inhibitor of CXCL12 binding to CXCR7. Gaussia luciferase bioluminescence was quantified as described in Figure 2, and data were presented as mean values ± SEM (n = 4 per condition). (B) Cocultures of CXCL12-CG/NG-CXCR4 or control CG/NG-CXCR4 cells were incubated for 2 hours with increasing concentrations of AMD3100 or CCX771, respectively, before measuring bioluminescence from Gaussia luciferase (n = 4 per condition). (C) Pairs of HeyA8 cells were incubated for 4 hours before adding increasing concentrations of AMD3100 for an additional 4 hours before quantify bioluminescence (n = 4 per condition). *P < .05. **P < .01.

Figure 4

Imaging CXCL12-CXCR4 binding in living mice with disseminated intraperitoneal ovarian cancer. (A) Pairs of HeyA8 ovarian cancer cells (CG/NG-CXCR4) or (CXCL12-CG/NG-CXCR4) were injected intraperitoneally into mice (1.25 x 10⁵ of each cell type per mouse). Two weeks after injection, mice were imaged with coelenterazine to detect bioluminescence from Gaussia luciferase complementation in cells with the interacting pair (CXCL12-CG/NG-CXCR4) or the control pair (CG/NG-CXCR4). Firefly luciferase images show relative numbers of HeyA8-NG-CXCR4 cells, which constitutively express this imaging reporter. Scale bars for imaging data depict ranges of pseudocolors with red and blue representing highest and lowest photon flux values, respectively. (B) Quantified data for Gaussia luciferase bioluminescence (n = 5 per group). Data were graphed as mean values ± SEM. *P < .05.

Figure 5

AMD3100 blocks CXCL12-CXCR4 binding in vivo. (A) Mice were injected intraperitoneally with 2.5 x 10⁵ each of HeyA8-CXCL12-CG and NG-CXCR4 cells. Baseline images for Gaussia luciferase complementation and firefly luciferase expressed constitutively by NG-CXCR4 cells were obtained 7 days after injecting ovarian cancer cells. Mice then were treated for 3 days with AMD3100 or vehicle control before repeating imaging studies. Representative images for mice treated with AMD3100 or vehicle control are displayed. Scale bars denote pseudocolor displays for bioluminescence for Gaussia and firefly luciferase images, respectively. (B) Quantified data for Gaussia luciferase (GL) activity (n = 7–8 per group). Change in bioluminescence from posttreatment to pretreatment values was calculated for each mouse, and mean values ± SEM were presented. *P < .05.

Figure 6

AMD3100 reduces tumor growth and prolongs survival of mice with metastatic ovarian cancer. (A) Mice were injected intraperitoneally with 1.25 x 10⁵ cells each of HeyA8-CXCL12-CG and NG-CXCR4 cells. After 10 days of tumor growth, mice were imaged for baseline Gaussia luciferase activity immediately before implantation of osmotic infusion pumps with AMD3100 or vehicle control. Repeat imaging studies for Gaussia luciferase complementation were performed on days 5 and 11 of treatment. The fold change in Gaussia luciferase bioluminescence relative to the pretreatment baseline value was determined for each mouse, and mean values for each group ± SEM were graphed. (B) Representative firefly luciferase images for relative numbers of HeyA8-NG-CXCR4 cells before and on days 5 and 11 of treatment with AMD3100 or vehicle control. Scale bar shows pseudocolor display for photon flux values on all images. (C) Quantified data for tumor growth measured by firefly luciferase activity. Fold change in bioluminescence on days 5 and 11 of treatment was determined relative to pretreatment values for each mouse, and data were presented as mean values ± SEM (n = 8 per group). (D) Kaplan-Meier survival curve for mice treated with AMD3100 or vehicle control. Black arrow denotes start of therapy. *P < .05. **P < .01.