Host Wnt5a Potentiates Microenvironmental Regulation of Ovarian Cancer Metastasis

Abstract

The noncanonical Wnt ligand Wnt5a is found in high concentrations in ascites of women with ovarian cancer. In this study, we elucidated the role of Wnt5a in ovarian cancer metastasis. Wnt5a promoted ovarian tumor cell adhesion to peritoneal mesothelial cells as well as migration and invasion, leading to colonization of peritoneal explants. Host components of the ovarian tumor microenvironment, notably peritoneal mesothelial cells and visceral adipose, secreted Wnt5a. Conditional knockout of host WNT5A significantly reduced peritoneal metastatic tumor burden. Tumors formed in WNT5A knockout mice had elevated cytotoxic T cells, increased M1 macrophages, and decreased M2 macrophages, indicating that host Wnt5a promotes an immunosuppressive microenvironment. The Src family kinase Fgr was identified as a downstream effector of Wnt5a. These results highlight a previously unreported role for host-expressed Wnt5a in ovarian cancer metastasis and suggest Fgr as a novel target for inhibition of ovarian cancer metastatic progression.Significance: This study establishes host-derived Wnt5a, expressed by peritoneal mesothelial cells and adipocytes, as a primary regulator of ovarian cancer intraperitoneal metastatic dissemination and identifies Fgr kinase as novel target for inhibition of metastasis.

Figure 1.

Wnt5a induces prometastatic ovarian cancer cell phenotypes. A, Diagram of mesomimetic culture comprised of three-dimensional type I collagen (RTC I) gels overlaid with LP9 human peritoneal MC (20). B, RFP-tagged OVCAR5 and OVCAR8 cells were treated with SFM or recombinant Wnt5a protein (rWnt5a; 0.4 μg/mL, 24 hours) as indicated. Cells from each treatment condition (5 × 10⁴) were incubated with mesomimetic cultures for 20 minutes or 1 hour, respectively, prior to washing and imaging. C, Quantification of adherent cells. D, Diagram of murine ex vivo peritoneal explant culture. Explants were pinned mesothelium-up on optically clear silastic resin. E, RFP-tagged OVCAR5 and OVCAR8 cells were treated with SFM or rWnt5a (0.4 μg/mL, 24 hours) as indicated prior to incubation with murine peritoneal explants in an ex vivo adhesion assay for 30 or 90 minutes, respectively, prior to washing and imaging. F, Quantification of adherent cells. Images of adherent cells were obtained using Echo Revolve fluorescent microscope at ×20 magnification. G, OVCAR5 and OVCAR8 cells were added to transwell migration chambers containing SFM or rWnt5a (0.4 μg/mL) and incubated for 12 and 18 hours, respectively. Migrated cells were fixed and stained with Diff-Quik Kit. H, Quantification of migrated cells. I, OVCAR5 and OVCAR8 cells were added to transwell invasion chambers containing type I collagen (100 μL of 200 μg/mL) and SFM or rWnt5a (0.4 μg/mL, 24 hours). Invaded cells were fixed and stained with Diff-Quik Kit. J, Quantification of invaded cells. Images of migrated and invaded cells were obtained using Olympus BH-42 microscope. Scale bar, 20 μm. All experiments in A–J were performed as triplicates, with three independent biological replicates per cell line. All results are presented as mean ± SEM and P values were calculated using a Student two-tailed t test. P < 0.05 is statistically significant. K, OVCAR5 and OVCAR8 cells were serum-starved overnight, then resuspended in SFM or rWnt5a (0.4 μg/mL) and cultured atop collagen I-coated coverslips for 24 hours. Cells were fixed with 4% PFA buffer and stained with Phalloidin 488 and DAPI. Cells were imaged with Leica DM5500 fluorescence microscope at ×25 magnification. Scale bar, 100 μm. L, OVCAR5 and OVCAR8 cells were incubated in SFM or rWnt5a (0.4 μg/mL) for 24 hours. Cells were added to murine peritoneal explants for 30 and 90 minutes, respectively. The peritoneal explants were fixed and processed for imaging by FEI-Magellan 400 Field Emission scanning electron microscope. Scale bar, 5, 10 μm.

Figure 2.

Wnt5a derived from peritoneal mesothelial cells alters ovarian cancer cell behavior. A–D, Relative expression of WNT5A by qRT-PCR. A, Comparison of expression levels between the human ovarian cancer cell line OVCAR3, the human peritoneal mesothelial cell line LP9, and primary human peritoneal mesothelial cells from two different donors (HPMC I and HPMC II). B, Comparison of expression levels between the murine ovarian cancer cell line ID8-Trp53^−/− and primary murine mesothelial cells (MPMC). C, Quantitation of Wnt5a protein level. The concentration of Wnt5a was measured in serum-free conditioned media from OVCAR3, LP9, and HPMC cells using ELISA according to the manufacturer’s specifications. D, Comparison of expression between the murine ovarian cancer cell line ID8-Trp53^−/− and murine visceral adipose obtained from periovarian fat (Ov-fat), periuterine fat (uter-fat), or omentum. All experiments in A–D were performed using n = 3 independent biological replicates per experiment. Results are presented as mean ± SEM, and statistical significance was calculated using a Student two-tailed t test. P < 0.05 is statistically significant. E, IHC analysis of Wnt5a in murine ovarian tissue, uterine fat, peritoneum, periovarian fat, and omentum, as indicated. Tissues were incubated with anti-Wnt5a (1:200 dilution), followed by a peroxidase-conjugated anti–rabbit-IgG and peroxidase detection using DAB as described in Materials and Methods. Images were acquired with Olympus BH-42 microscope. Scale bar, 50 and 20 μm. Arrowheads, example areas of positive staining. F, Quantification of ovarian cancer cell adhesion to mesomimetic cultures. RFP-tagged OVCAR5 and OVCAR8 cells were serum-starved overnight, treated with conditioned medium from control human peritoneal MC (LP9-CND) or from LP9 cells in which WNT5A is silenced (LP9-Wnt5a^KD-CND) for 24 hours, then allowed to adhere to a mesomimetic culture 20 minutes and 1 hour, respectively. G, Quantification of ovarian cancer cell adhesion to murine peritoneal explants. RFP-tagged OVCAR5 and OVCAR8 cells were treated with either LP9-CND or LP9-Wnt5a^KD-CND for 24 hours prior to incubation with murine peritoneal explants in an ex vivo adhesion assay for 30 and 90 minutes, respectively. H, Analysis of migration. OVCAR5 and OVCAR8 cells were serum-starved overnight, then, cells were added to transwell migration chambers containing LP9-CND or LP9-Wnt5a^KD-CND and incubated for 12 and 18 hours, respectively. Migrated cells were fixed and stained with Diff-Quik Kit and quantified using ImageJ. I, Analysis of invasion. OVCAR5 and OVCAR8 cells were serum-starved overnight, then, cells were added to transwell invasion chambers containing type I collagen gels (100 μL of 200 μg/mL) with LP9-CND or LP9-Wnt5a^KD-CND and incubated for 24 hours. Invaded cells were fixed and stained with Diff-Quik Kit and quantified using ImageJ. All experiments were done in triplicate, with three independent biological replicates per each cell line. All results are presented as mean ± SEM and P-values were calculated using a Student two-tailed t test. P < 0.05 is statistically significant. NS, nonsignificant; *, P < 0.05; **, P < 0.01.

Figure 3.

Conditional silencing of host WNT5A reduces overall peritoneal metastatic burden. A, Overview of breeding strategy and tamoxifen (TMX) treatment to generate WNT5A knockout mice. B, Summary of murine cohorts used in the allograft tumor study. C, Mice were injected intraperitoneally with 5 × 10⁶ RFP-tagged ID8-Trp53^−/− syngeneic murine ovarian cancer cells. Beginning at 3 weeks postinjection, tumor burden in situ was longitudinally imaged weekly using a Bruker Xtreme In Vivo Imaging system. Mice were sacrificed at 5 weeks postinjection and each abdominal cavity was exposed and imaged using the Bruker Xtreme In Vivo Imaging system. E, Quantification of abdominal tumor burden area by dividing the tumor area by the scale-adjusted body weight of each mouse. E, Abdominal tumor intensity was calculated by dividing the tumor intensity by the scale-adjusted body weight of each mouse. Quantification was performed using ImageJ. All results are presented as means ± SEM and P values were calculated using a Student two-tailed t test. P < 0.05 is statistically significant. F, Representative organ-specific tumor burden images for each cohort. Individual organs were dissected and imaged ex vivo using the Bruker Xtreme In Vivo Imaging system. G, Quantification of organ-specific tumor burden. The “organ area fraction” was quantified by dividing the tumor area by the adjusted organ weight for all mice in each cohort. H, All mice were subjected to peritoneal lavage with 2 mL of PBS, then, ascites/PBS fluid was collected. Ascites volume was calculated by subtracting 2 from the total volume. All results are presented as mean ± SEM and P values were calculated using a Student two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, with P < 0.05 being statistically significant.

Figure 4.

CyTOF analysis of immune profiles in peritoneal lavage. Peritoneal lavage was collected from tumor-free Wnt5a^KO and control mice (n = 3/genotype) as described in Materials and Methods and labeled with metal-conjugated antibodies for mass cytometry analysis as described previously. A, Gating strategy for CyTOF data. B, Frequencies of indicated immune populations of all CD45⁺ immune cells and PD-1 expression in indicated T-cell subsets in control (Ctrl) versus Wnt5a^KO (KO) hosts. NS, data do not reach statistical significance (P > 0.05).

Figure 5.

Tumor-bearing WNT5A knockout mice have altered peritoneal cytokine profiles. A, Peritoneal lavage obtained from mice with conditional WNT5A knockout or controls was analyzed using the murine Proteome Profiler Cytokine Array according to the manufacturer’s specifications. Spots showing differential expression are boxed.B, Each cytokine has duplicate spots on the blot and each spot intensity was quantified by measuring pixel density using ImageJ following the manufacturer’s protocol. All results are presented as mean ± SEM.

Figure 6.

Silencing of host WNT5A alters the immune landscape of peritoneal metastases. A, Immunofluorescence staining of infiltrating FOXP3⁺ T cells (red) and CD8⁺ T cells (green) in peritoneal metastases at magnification ×20. B, The ratio of average of CD8⁺ and FOXP3⁺ cell counts for three mice from each cohort was used for statistical analysis. Cell counting was done manually in ImageJ. C, Immunofluorescence staining of infiltrating F4/80⁺ (red) iNOS⁺ (green) M1 macrophages in peritoneal metastases. E, Immunofluorescence staining of infiltrating F4/80⁺ (red) CD206⁺ (green) M2 macrophages in peritoneal metastases. D–F, For macrophage quantification, 10 independent areas with the most abundant macrophages per mouse were selected and imaged with 20 objective lens and area percentage covered by F4/80⁺ iNOS⁺ or F4/80⁺ CD206⁺ cells was calculated per each image using ImageJ. A representative image is shown. Imaging was done with Leica DM5500 fluorescence microscope. All results are presented as means ± SEM, and P values were calculated using a Student two-tailed t test. P < 0.05 is statistically significant.

Figure 7.

Identification of Fgr as a downstream mediator of Wnt5a prometastatic cellular behavior. A, OVCAR5 and OVCAR8 cells were treated with SFM or rWnt5a protein (0.4 μg/mL, 24 hours) prior to lysis. Lysates were analyzed using the Proteome Profiler Human Phospho-Kinase Array according to the manufacturer’s specifications. Log₂-fold change of phosphorylated proteins in the phosphokinase array is shown. B, Quantification of Fgr (Y412) phosphorylation by measuring pixel density for duplicate spots on the phospho-array blot using ImageJ. Data are presented as mean ± SEM. C, Validation of Fgr expression and phosphorylation. OVCAR5 and OVCAR8 cells were serum-starved for 2 hours then incubated with the Fgr inhibitor TL02–59 (0.01 μmol/L) for 2.5 hours. After washing, cells in SFM were incubated with rWnt5a protein (0.4 μg/mL) for the time points indicated. Ctrl, cells without rWnt5a treatment. Lysates were electrophoresed on SDS-polyacrylamide gels and immunoblotted with the antibodies noted. One representative blot from three independent biological replicates is shown. D and E, RFP-tagged OVCAR5 and OVCAR8 cells were treated with SFM, rWnt5a (0.4 mg/mL), rWnt5a (0.4 μg/mL), and the specific Fgr kinase inhibitor TL02–59 (1 μmol/L) or TL02–59 (1 μmol/L) for 24 hours, as indicated, prior to incubation with mesomimetic culture for 20 minutes or 1 hour (D), respectively, or with murine peritoneal explants in an ex vivo adhesion assay for 30 or 90 minutes (E), respectively. Quantification of adherent cells is shown for each condition. F, OVCAR5 and OVCAR8 cells were added to transwell migration chambers containing SFM, rWnt5a (0.4 μg/mL), rWnt5a (0.4 μg/mL), and TL02–59 (1 μmol/L), or TL02–59 (1 μmol/L) and incubated for 12 and 18 hours, respectively. Quantification of migrated cells in each condition.