Monocyte/macrophage and T-cell infiltrates in peritoneum of patients with ovarian cancer or benign pelvic disease

Abstract

Background: We previously showed that tumor-free peritoneum of patients with epithelial ovarian cancer (EOC) exhibited enhanced expression of several inflammatory response genes compared to peritoneum of benign disease. Here, we examined peritoneal inflammatory cell patterns to determine their concordance with selected enhanced genes.

Methods: Expression patterns of selected inflammatory genes were mined from our previously published data base. Bilateral pelvic peritoneal and subjacent stromal specimens were obtained from 20 women with EOC and 7 women with benign pelvic conditions. Sections were first stained by indirect immunoperoxidase and numbers of monocytes/macrophages (MO/MA), T cells, B cells, and NK cells counted. Proportions of CD68+ cells and CD3+ cells that coexpressed MO/MA differentiation factors (CD163, CCR1, CXCR8, VCAM1, and phosphorylated cytosolic phospholipase A2 [pcPLA2]), which had demonstrated expression in EOC peritoneal samples, were determined by multicolor immunofluorescence.

Results: MO/MA were present on both sides of the pelvic peritoneum in EOC patients, with infiltration of the subjacent stroma and mesothelium. CD68+ MO/MA, the most commonly represented population, and CD3+ T cells were present more often in EOC than in benign pelvic tumors. NK cells, B cells, and granulocytes were rare. CXCL8 (IL-8) and the chemokine receptor CCR1 were coexpressed more frequently on MO/MA than on CD3+ cells contrasting with CD68+/CD163+ cells that coexpressed CXCL8 less often. An important activated enzyme in the eicosanoid pathway, pcPLA2, was highly expressed on both CD68+ and CD163+ cells. The adherence molecule Vascular Cell Adhesion Molecule-1 (VCAM1) was expressed on CD31+ endothelial cells and on a proportion of CD68+ MO/MA but rarely on CD3+ cells.

Conclusion: The pelvic peritoneum in EOC exhibits a general pattern of chronic inflammation, represented primarily by differentiated MO/MA, and distinct from that in benign conditions concordant with previous profiling results.

Figure 1

Selected genes expressed at different levels in the peritoneum and stroma of patients with EOC vs in patients with benign pelvic disease. The red bars indicate the malignant phenotype and the blue bars the benign controls. The significance level of each gene expression between benign and malignant phenotypes is presented as P_(t2) values.

Figure 2

MO/MA & T-cell infiltration in peritoneum. Left upper shows peritoneum of patient w/benign fibrothecoma with scant LCA+ leukocytes below the single layer of mesothelium. Remaining 5 panels show tumor-free peritoneum from a patient with EOC. Upper middle shows large number of LCA+ cells; upper right shows large number of CD68+ cells; lower left shows relatively fewer CD3+ cells; lower middle shows negative isotype control; lower right shows H&E. Magnification---200×

Figure 3

Triple immunofluorescence costaining of frozen right peritoneal tissues were stained with CD68 (red), CD31 (blue), and keratin (CK) (green) antibodies (Rows 1 & 2) Row 1, peritoneal cells from a patient with EOC (ID 266 m) appear yellow from the colocalization of CD68 (red) and CK (green) on some surface mesothelial cells. CD31 staining (blue) indicates endothelial cells just under the mesothelium. Row 2, peritoneal cells from a patient with benign cystic teratoma of the ovary (ID 283b) show prominent staining for keratin in the single cell mesothelial layer but no staining for CD68 staining (red) and positive staining for endothelial cells (blue). Rows 3 and 4, peritoneal cells from patient ID#235 showed colocalization of CD68 (blue) and CD163 (green) appearing cyan color; CD68 (blue) and CXCL8 (red) costaining showed magenta effect and no color changed in CD163+ cells (green). Images were analyzed by confocal laser scanning microscopy (magnification 400×). H&E stained sections are shown for comparison.

Figure 4

Stained cytospin preps of mononuclear leukocytes isolated from ascitic fluid on F:H density cushion (patient ID 290; top row) or from frozen tissue (patient ID 235; bottom row) were fixed with 4% paraformaldehyde and double-stained with phospho-cPLA2 (Ser505) antibody (red) and CD68 (green) or CD163 (green). Indirect immunofluorescence showed pcPLA2 in both the nucleus and cytoplasm, and results were similar in the cells from ascites and those from tissue. Cells costained for CD68 and cPLA2 showed colocalization of both markers in the cytoplasm and in the nucleus (yellow). However, in CD163+ cells, cPLA2 was seen only in relation to the nucleus.