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. 2015 Nov 15;75(22):4742-52.
doi: 10.1158/0008-5472.CAN-14-3373. Epub 2015 Oct 15.

Macrophage Blockade Using CSF1R Inhibitors Reverses the Vascular Leakage Underlying Malignant Ascites in Late-Stage Epithelial Ovarian Cancer

Diana L Moughon  1 , Huanhuan He  2 , Shiruyeh Schokrpur  1 , Ziyue Karen Jiang  3 , Madeeha Yaqoob  4 , John David  5 , Crystal Lin  1 , M Luisa Iruela-Arispe  6 , Oliver Dorigo  2 , Lily Wu  7
Affiliations

Macrophage Blockade Using CSF1R Inhibitors Reverses the Vascular Leakage Underlying Malignant Ascites in Late-Stage Epithelial Ovarian Cancer

Diana L Moughon et al. Cancer Res. .

Abstract

Malignant ascites is a common complication in the late stages of epithelial ovarian cancer (EOC) that greatly diminishes the quality of life of patients. Malignant ascites is a known consequence of vascular dysfunction, but current approved treatments are not effective in preventing fluid accumulation. In this study, we investigated an alternative strategy of targeting macrophage functions to reverse the vascular pathology of malignant ascites using fluid from human patients and an immunocompetent murine model (ID8) of EOC that mirrors human disease by developing progressive vascular disorganization and leakiness culminating in massive ascites. We demonstrate that the macrophage content in ascites fluid from human patients and the ID8 model directly correlates with vascular permeability. To further substantiate macrophages' role in the pathogenesis of malignant ascites, we blocked macrophage function in ID8 mice using a colony-stimulating factor 1 receptor kinase inhibitor (GW2580). Administration of GW2580 in the late stages of disease resulted in reduced infiltration of protumorigenic (M2) macrophages and dramatically decreased ascites volume. Moreover, the disorganized peritoneal vasculature became normalized and sera from GW2580-treated ascites protected against endothelial permeability. Therefore, our findings suggest that macrophage-targeted treatment may be a promising strategy toward a safe and effective means to control malignant ascites of EOC.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Murine ID8 epithelial ovarian cancer model. A, bioluminescent imaging of three representative animals bearing intraperitoneal Renilla luciferase-marked ID8 tumor cells at 9 and 12 weeks posttumor implantation (left images). Right graph shows the maximum signal intensity (radiance = p/sec/cm2/sr) in the peritoneal cavity (n = 5). B, proportion of specified cell types (CD45+ F4/80+ macrophages, CD4+ and CD8+ T cells, and GFP+ tumor cells) found floating in the ascites as measured by flow cytometry at week 10 and week 12 posttumor cell implantation. C, ratios of M2 macrophages (CD45+ F4/80+ MHCII−) to M1 macrophages (CD45+ F4/80+ MHCII+) and CD4+ T cells to CD8+ T cells in the ascites at week 10 and week 12 posttumor cell implantation. D, severe anemia is seen in late stages of intraperitoneal ID8 model (n = 3–4). *, P < 0.05; ***, P < 0.001.
Figure 2
Figure 2
Vascular deregulation in ID8 EOC. A, mesentery tissues isolated from the intestinal region were stained for vasculature (isolectin, red) and macrophages (CD11b in naïve or CD206 in tumor-bearing, green). B-D, vascular parameters such as vessel density, vessel width, and branch points, respectively, were scored in the mesentery tissues (n = 4). E, vasculature function assay with perfused lectin (red) and surface stained CD31 (green) assessed by whole mount. Quantification is the percentage of CD31+ vessels that are also lectin+ and normalized to naïve mouse vasculature (n = 2–4). Scale bars, 50 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3
Figure 3
CSF1R inhibition improves the health of mice-bearing ID8 EOC. A, ID8 cancer progression and GW2580 (GW) treatment timeline. B, CSF1R inhibition reduces ascites volume. Graph shows average ascites volume/animal (n = 9–10). C, hematocrit after 2 weeks of treatment (n = 4). D, content of macrophage in ascites before and after GW2580 treatment. Samples of ascites (<50 μL) drained immediately before or after a 2-week treatment course of vehicle or GW2580 were assessed for macrophage (CD45+, F4/80+) levels (n = 3). E, absolute numbers of total, M2 and M1 ascites macrophages with and without GW2580 treatment (n = 3–4). ns, nonsignificant. F, the M2:M1 macrophage ratios and CD4:CD8 T-cell ratios in control-treated and GW2580-treated ascites (n = 7–10). G, ascites macrophage CCR2 expression (n = 3). *, P < 0.05; **, P < 0.01.
Figure 4
Figure 4
CSF1R inhibition reduces vascular dysregulation in ID8 and OVCAR3 EOC. Mesentery vasculatures (isolectin, red) and macrophages (CD206+, green) in control and GW2580-treated ID8-bearing animals (A) and OVCAR3-bearing animals (E), as assessed by staining of whole mount tissue. Vascular parameters (vessel density, vessel width, branch points) in ID8-bearing (B-D) and OVCAR3-bearing (F-H) animals (n = 4). Scale bars, 50 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 5
Figure 5
CSF1R inhibition reduces vascular dysfunction in ID8 EOC. A, this graph shows five representative endothelial monolayers’ resistance plotted over 4 hours. B, four-hour endothelial layer permeability assay of control versus GW-treated cell-free ascites sera (n = 7). C, VE-Cadherin expression in EC monolayers treated with ascites sera. D, vasculature function assay with perfused lectin (red) and surface stained CD31 (green) assessed by whole mount. Quantification is the percentage of CD31+ vessels that are also lectin+ and normalized to naive mouse vasculature (n = 4). E, vascular leakage Miles assay that quantified amount of Evans Blue in mesentery (ng dye/mg tissue; n = 2–3). Scale bars, 50 μm.*, P < 0.05; **, P < 0.01.
Figure 6
Figure 6
EOC patient ascites and macrophages. A, EOC patient ascites cellular content is largely macrophages (CD33+ CD68+) and tumor cells (CD33− Muc-1+; n = 8). B, the macrophages in patient ascites are largely “M2” tumor-promoting macrophages (CD33+ CD68+ CD206+ HLA-DRO−) as opposed to the “M1” antitumor macrophages (CD33+ CD68+ CD206− HLA-DR+), and CD4+ T cells outnumber the CD8+ T cells (n = 6). C, graph of HUVEC endothelial layer resistance over 4 hours during a permeability assay with patient ascites serum. Beside each patient number are parentheses with the percent of macrophages in that patient’s ascites. D, the resistance at four hours (from C) correlated negatively with the macrophage content of the ascites (n = 5).
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