{"paper_id":"f8442bd1-fd10-4b88-8d13-f88d3a06f4b3","body_text":"BioMed Central\nPage 1 of 11\n(page number not for citation purposes)\nJournal of Translational Medicine\nOpen AccessResearch\nMonocyte/macrophage and T-cell infiltrates in peritoneum of \npatients with ovarian cancer or benign pelvic disease\nXipeng Wang1, Michael Deavers2, Rebecca Patenia3, Roland L Bassett Jr4, \nPeter Mueller4, Qing Ma5, Ena Wang6 and Ralph S Freedman*3\nAddress: 1Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Tiao Tong University, Shanghai, China, 2Department of Pathology, \nThe University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA, 3Department of Gynecologic Oncology, The University of Texas M. \nD. Anderson Cancer Center, Houston, Texas, USA, 4Department of Biostatistics and Applied Mathematics, The University of Texas M. D. Anderson \nCancer Center, Houston, Texas, USA, 5Department of Blood and Marrow Transplantation, The University of Texas M. D. Anderson Cancer Center, \nHouston, Texas, USA and 6Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland, USA\nEmail: Xipeng Wang - xipengwang@yahoo.com; Michael Deavers - mdeavers@mdanderson.org; Rebecca Patenia - rpatenia@mdanderson.org; \nRoland L Bassett - rlbasset@mdanderson.org; Peter Mueller - pm@odin.mdacc.tmc.edu; Qing Ma - qma@mdanderson.org; \nEna Wang - ewang@cc.nih.gov; Ralph S Freedman* - rfreedma@mdanderson.org\n* Corresponding author    \nAbstract\nBackground: We previously showed that tumor-free peritone um of patients with epithelial ovarian cancer\n(EOC) exhibited enhanced expression of several inflammatory response genes compared to peritoneum of benign\ndisease. Here, we examined peritoneal inflammatory cell patterns to determine their concordance with selected\nenhanced genes.\nMethods: Expression patterns of selected in flammatory genes were mined fr om our previously published data\nbase. Bilateral pelvic peritoneal and subjacent stromal specimens were obtained from 20 women with EOC and\n7 women with benign pelvic conditions. Sections were fi rst stained by indirect immunoperoxidase and numbers\nof monocytes/macrophages (MO/MA), T cells, B cells, and NK cells count ed. Proportions of CD68+ cells and\nCD3+ cells that coexpr essed MO/MA differ entiation factors (CD163, CCR1, CXCR8, VCAM1, and\nphosphorylated cytosolic phospholipase A 2 [pcPLA2]), which had demonstrated ex pression in EOC peritoneal\nsamples, were determined by multicolor immunofluorescence.\nResults: MO/MA were present on both sides of the pelvic peri toneum in EOC patients, with infiltration of the\nsubjacent stroma and mesothelium. CD68+ MO/MA, the most commonly represented population, and CD3+ T\ncells were present more often in EOC than in benign pelvic tumors. NK cells, B cells, and granulocytes were rare.\nCXCL8 (IL-8) and the chemokine receptor CCR1 were coexpressed more frequently on MO/MA than on CD3+\ncells contrasting with CD68+/CD163+ cells that coexpressed CXCL8 less often. An important activated enzyme\nin the eicosanoid pathway, pcPLA 2, was highly expressed on both CD 68+ and CD163+ cells. The adherence\nmolecule Vascular Cell Adhesion Molecule-1 (VCAM1 ) was expressed on CD31+ endothelial cells and on a\nproportion of CD68+ MO/MA but rarely on CD3+ cells.\nConclusion: The pelvic peritoneum in EOC e xhibits a general pattern of ch ronic inflammation, represented\nprimarily by differentiated MO/MA, and distinct from that in benign conditions concordant with previous profiling\nresults.\nPublished: 06 July 2006\nJournal of Translational Medicine 2006, 4:30 doi:10.1186/1479-5876-4-30\nReceived: 24 May 2006\nAccepted: 06 July 2006\nThis article is available from: http://www.translational-medicine.com/content/4/1/30\n© 2006 Wang et al; licensee BioMed Central Ltd.\nThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), \nwhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 2 of 11\n(page number not for citation purposes)\nBackground\nEpithelial ovarian cancer (EOC) results in 5 year survival\nrates of only 25–30% for patients with stage III and IV dis-\nease [1], contrasting with the 90% survival rates of\npatients with stage I disease, where notably peritoneal and\nserosal disease is absent. It is perhaps a paradox that the\nperitoneum which is organized to protect the integrity of\nintraabdominal organs by facilitating infiltration of\ninflammatory cells to sites of injury and infection, might\nalso serve to facilitate the promotion of tumor growth and\nspread.\nAs EOC advances and penetrates the capsular layer of the\novary, it also carries the potential to expose the peritoneal\nsurface to tumor-cell secreted products. The peritoneum\nand its extension, the intestinal serosa, include a vast sur-\nface area for transit of inflammatory cells into the abdom-\ninal cavity. Its surface mesothelium and submesothelial\nstroma and structure pose no substantial barriers to\ninflammatory modulatory cytokines, chemokines and\nother molecules produced by the tumor or its metastasis,\nat least to a depth of approximately 1 mm [2]. The stroma\nconsists of a collagen-based matrix, blood vessels, lym-\nphatics, nerve fibers, and rare hematogenous cells [3,4].\nSurgery for EOC often reveals changes in the non-tumor-\nbearing peritoneum such as thickening or edema,\nenhanced vascular patterns, and soft or firm adhesions\n[5]. The peritoneum and intestinal serosa may have a\nflorid appearance similar to that found in peritonitis.\nDespite this evidence of inflammation, the inflammatory\nprocess in the peritoneum of patients with EOC has not\nbeen adequately described or characterized.\nUsing a previously validated cDNA microarray platform\nconsisting of 17,500 clones enriched with inflammatory\nand immunologically relevant genes [6-8], we previously\nshowed that the gene profiles of the pelvic peritoneum in\npatients with EOC exhibited a pattern consistent with the\npresence of MO/MA differentiation, activation, and cell\nsurvival and that the pattern was different from that of the\nperitoneum of patients without cancer or that of the\ntumor itself [9]. Categorizing genes on the basis of anno-\ntated gene function led to our observing that genes associ-\nated with inflammation were overexpressed in non-tumor\nbearing peritoneum of patients with ovarian cancer as\ncompared with the peritoneum of patients with benign\novarian tumors.\nThe purpose of the study reported here was to describe the\nglobal pattern of the main inflammatory cell populations\nin the peritoneum and stroma and to determine whether\nthe magnitude of expression of a limited group of inflam-\nmatory genes could be confirmed at the cellular pro-\nteomic level in peritoneal tissue and ascites cells.\nMethods\nPeritoneal and subjacent stromal biopsy specimens were\nobtained from 20 patients with EOC and from 7 patients\nwith benign ovarian or other pelvic tumors who under-\nwent surgery at M. D. Anderson Cancer Center according\nto a protocol approved by the appropriate institutional\nreview board. Demographic characteristics of those\npatients are shown in Table 1. Biopsy samples were\nobtained from the peritoneum and from the submesothe-\nlial stroma on both sides of the pelvis, approximately 2\ncm from the nearest visible tumor deposits, as quickly as\npossible after the abdominal cavity was accessed. Perito-\nneal biopsy samples were obtained carefully without prior\nmanipulation of the chosen biopsy sites to minimize arti-\nfact induced variability. As controls, specimens were\nobtained from similar peritoneal sites in consenting sub-\njects who were undergoing pelvic abdominal surgery but\nwho did not have a diagnosis of cancer. The combined\nthickness of the peritoneal and separately obtained deeper\nstromal biopsy specimens was estimated at 1–2 millime-\nters. A technician was present in the operating room to\nreceive and process all biopsy specimens. All specimens\nwere bisected. One portion, for histopathologic, immu-\nnohistochemical, and immunofluorescence costaining,\nwas collected and transported to the lab on ice where it\nwas snap-frozen in Polyfreeze Tissue Freezing Medium\n(Polysciences, Warrington, PA). Another portion, to be\nused for microarray, was placed in a sterile tube contain-\ning 5% dextrose 0.2% sodium chloride solution and\ntransported on ice to the laboratory. The tissue was\nremoved from the saline solution and snap-frozen in a\nvial with RNAlater (Ambion, Austin, TX) to minimize\nRNA metabolism and degradation. Subsequently, perito-\nneal tissue was also obtained from several additional\npatients for eicosanoid studies. This tissue was placed dry\ninto a sterile tube and snap-frozen in liquid nitrogen in\nthe operating room. All tissues were stored at -80°C.\nBenign cases included: ovarian fibrothecoma (3), serous\ncystoadenoma or cystoadenofibroma (3), and ovarian\npapillary proliferation (1). A gynecologic pathologist\n(M.D.) reviewed all hematoxylin-and-eosin (H&E) -\nstained sections from specimens used in this study. Peri-\ntoneal specimens showing microscopic tumor involve-\nment were not included in the studies described here.\nImmunohistochemical staining of peritoneal biopsy tissues\nIndirect immunoperoxidase (IIP) staining\nTo determine the proportions of infiltrating mononuclear\nleukocyte populations in the peritoneal stroma, cryopre-\nserved peritoneal biopsy specimens were cut and stained,\nusing an avidin-biotin immunoperoxidase method\n[10,11]. IIP staining is generally considered more sensitive\nand specific than H&E for staining and identifying mono-\nnuclear leukocyte populations in cryopreserved tissue.\nBriefly, 6-μm sections of cryopreserved peritoneal tissues\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 3 of 11\n(page number not for citation purposes)\nwere immediately fixed with acetone for 10 minutes, air-\ndried for 30 minutes, and then kept at 20°C overnight.\nSections were then air-dried for another 30 minutes at\nroom temperature and endogenous peroxidase activity\nwas blocked by incubation in 0.3% H 2O2 in PBS for 15\nminutes. Sections were then washed three times in PBS,\nand nonspecific reactions were blocked with 2% normal\nhorse serum for 30 minutes. Sections were then incubated\nfor 2 hours at room temperature with the primary anti-\nbodies as follows. For the immunohistochemical analy-\nses, primary antibodies were: Mouse anti-human CD45\nleukocyte common antigen [LCA] clones 2B11 + PD7/26,\ncatalog no. M0701, Mo IgG1, 1/400 (DakoCytomation,\nCarpinteria, CA); mouse anti-human CD3 clone T3-4B5,\ncatalog no. M0756, Mo IgG1, 1/225 (DakoCytomation);\nmouse monoclonal antibody KP1 to CD68, catalog no.\nab955, Mo IgG1, 1/2000 (Abcam, Cambridge, MA);\nmouse anti-SCLC (CD56, N-CAM) clone 123C3. Mo\nIgG1, 1/50 (Zymed Labs, San Francisco, CA); and mouse\nanti-human CD20 clone B-LyI, catalog no. M0774, Mo\nIgG1 kappa 1/700 (DakoCytomation). Secondary anti-\nbodies used were: biotinylated horse anti-mouse IgG\n(1:200) (Vector Laboratories, Burlingame, CA); Universal\nLSAB kit/HRP Rabbit/Mouse, catalog no. K0675 (Dako-\nCytomation), and appropriate isotype controls. Optimal\nconditions for staining with each antibody were deter-\nmined by using appropriate test tissues. After being\nwashed with PBS three times, sections were incubated\nwith the appropriate secondary antibody for 1 hour at\nroom temperature. Sections were then washed again with\nPBS and incubated with avidin-biotin peroxidase conju-\ngate (ABC Kit, Vector Laboratories, catalog no. PK6102) at\na dilution of 1:100 for 30 minutes at room temperature,\nafter which AEC substrate (Vector Laboratories, catalog\nno. SK-4200) was added for 10 minutes. Sections were\nwashed with tap water, counterstained with Vector hema-\ntoxylin (Vector Laboratories) for 1 minute, and mounted\nwith permanent aqueous mounting medium (Biomeda,\nFoster City, CA, catalog no. M03) [10].\nCoded slides for indirect immunoperoxidase staining\nwere counted in nine areas per tissue section by random\nfield selection, and the number of cells was averaged per\n0.08 mm2. Distributions and ratios of MO/MA and T cells\nwere determined by quantitative immunochemical analy-\nsis with a Leica DM LB (Leica, Germany) image analyzer\nequipped with Image Pro Plus software (Media Cybernet-\nics, Silver Springs, MD) [9]. The Image ProPlus software\nprogram evaluates random counting of positive cells by\nusing a grid mask in the process menu, and artifacts are\nremoved by using the delete option.\nMulti antibody immunofluorescence costaining and confocal \nmicroscopy of peritoneal biopsy specimens\nIn situ cell populations or subsets were examined by mul-\nticolor immunofluorescence costaining to detect surface\nreceptors and certain cytoplasmic proteins that had been\nidentified in our previous transcriptome studies [9,12].\nFor the experiments described here, CCR1, CXCL8 (IL-8),\nCD163, and VCAM1 were included for costaining mono-\nTable 1: Clinical characteristics of the 20 chemo-naive patients with epithelial ovarian cancer\nCharacteristics N (%)\nMean Age, years (range)\nPatients with malignant disease (n = 20) 60 (36 – 79)\nPatients with benign ovarian disease (n = 7) 64 (47 – 83)\nEOC Histology\nSerous 10 (50%)\nMucinous 2 (10%)\nEndometrioid 2 (10%)\nClear cell 1 (5%)\nMixed 5(25%)\nDisease Stage\nI – II 2 (10%)\nIII – IV 18 (90%)\nTumor Grade\nI 2 (10%)\nII 2 (10%)\nIII 16 (80%)\nSurgical Debulking\nOptimal 11 (55%)\nSuboptimal 9 (45%)\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 4 of 11\n(page number not for citation purposes)\nnuclear leukocytes that expressed CD68+ or CD3+. In\nsome experiments, MO/MA and the CD163+ subset were\ncostained with phosphorylated cytosolic phospholipase 2\n(cPLA2), which together with sPLA2 (Group 2a) was over-\nexpressed in peritoneal samples from EOC patients.\nFreshly cut tissues (6 μm) were fixed in 4% paraformalde-\nhyde for 20 minutes at room temperature, after which sec-\ntions were washed in PBS, permeabilized with 0.5%\nTriton 100×, blocked with 5% normal goat serum, and\nincubated with primary antibodies overnight at 4°C.\nIIF triple costaining\nA sequential staining technique was used for this method\nas follows: 3 hours incubation with the first primary anti-\nbody (red) at RT, overnight incubation with the second\nprimary antibody (blue) at 4°C, and 3 hours incubation\nwith the third primary antibody at RT. Secondary antibod-\nies were incubated with the sections for 1 hour after the\nincubations with the primary antibodies were complete.\nNonspecific binding was blocked by adding 5% normal\ngoat serum for 1 hour. The primary antibodies used were:\nmouse anti-human CD3 clone T3-4B5, Mo IgG1 kappa,\n1:225 dilution, catalog no. M0756 (DakoCytomation);\npolyclonal rabbit anti-human CD3, 1:100, catalog no.\nA0452 (DakoCytomation); mouse anti-human CD68,\nMo IgG2a, 1:30, catalog no. MCA1815 (Serotec, Raleigh,\nNC); mouse monoclonal antibody KP1 to CD68, Mo\nIgG1 kappa, 1:1500, catalog no. ab955 (Abcam); mouse\nanti-human CD163, Mo IgG1, 1:100, catalog no.\nMCA1853 (Serotec); mouse anti-humanVCAM1 clone\n1.4C3, Mo IgG1 kappa, 1:50, catalog no. M7106 (Dako-\nCytomation); mouse anti-human CCR1, Mo IgG2B,\n1:100, catalog no. MAB145 (R&D Systems, Minneapolis,\nMN); mouse anti-human CD14, Mo IgG2a kappa, catalog\nno. M0825 (DakoCytomation); polyclonal rabbit anti-\nhuman IL-8, 1:5, catalog no. AHC0881 (Biosource,\nCamarillo, CA); phospho-cPLA2 (Ser 505) antibody (rab-\nbit) #2831, 1:50 (Cell Signaling Technology, Danvers,\nMA); mouse anti-human CD31 (PECAM-1, Platelet gpIIa\nMolecule), catalog no. C2383-02, Mo IgG2b, 1:100\n(United States Biological, Swampscott, MA); and mouse\nanti-human cytokeratin clone AE1/AE3, Mo IgG1 kappa,\n1:50, catalog no. M3515 (DakoCytomation). The second-\nary antibodies used depended on the isotype of the pri-\nmary antibodies, and included Cy2-conjugated (green)\nAffiniPure goat anti-mouse IgG, Fc γ subclass 1-specific,\ncatalog no. 115–225-205; Cy3-conjugated (red) Affin-\niPure goat anti-rabbit IgG (H+L), catalog no. 111–165-\n144; Cy5-conjugated (blue) AffiniPure goat anti-mouse\nIgG, Fc γ subclass 2b-specific, catalog no. 115–175–207;\nCy5-conjugated (blue) AffiniPure goat anti-mouse IgG,\nFcγ subclass 2a-specific, catalog no. 115–175–206; Cy3-\nconjugated (red) AffiniPure goat anti-mouse IgG, Fcγ sub-\nclass 2a-specific (minimal cross-reaction with bovine, and\nrabbit serum proteins), catalog no. 115–165–206 (all\nfrom Jackson ImmunoResearch Laboratories, West Grove,\nPA). Negative controls employed secondary antibodies\nalone.\nTissue sections were mounted with Slow-Fade Gold Anti-\nFade reagent (catalog no. S36936, Molecular Probes) and\nviewed with a Olympus FV500 laser scanning confocal\nmicroscope; images were captured at 400× and 600× mag-\nnification by Fluoview software Version 4.3.\nStatistics\nWilcoxon tests were used to compare the distribution of\nmarkers between malignant and benign samples. Paired t-\ntests were used to compare inflammatory cell numbers\nfrom each side of the pelvis and between the superficial\nand deeper stroma.\nResults and discussion\nWe first examined our data base of more than 50 inflam-\nmation-linked genes that have been shown to be\nexpressed differently in malignant and benign perito-\nneum [9]. The genes in Figure 1 were mined from a data-\nbase that supported our earlier report on the peritoneal\ntranscriptome [9]. Gene expression levels were generated\nby centering followed by cluster analysis and displayed as\ndendrograms (trees) [13]. Here we show expression of\nseveral of these genes that encode for the following mole-\ncules: CXCL8, a contributor to tumor angiogenesis and\nleukocyte chemotaxis, CD163 the scavenger molecule\nassociated with MA differentiation, CCR1, a chemokine\nreceptor expressed on different leukocytes, including MA\nand that binds to multiple CC ligands produced by ovar-\nian cancer cells, and MA, VCAM1, an adherence moledule\nand ligand for VLA-4 integrin and induced on endothelial\ncells by tumor necrosis factor (TNF α ), interleukin 1 α\n(IL1α ) and certain other cytokines. We also detected\nincreased expression of phosphorylated phospholipase\nA2 (sPLA2), the activated cytosolic form of the protein\nthat releases arachidonic acid from membranes (a critical\nearly step in the eicosanoid pathway) and that can induce\nCXCL8 (IL-8), IL-6, and CD44 production. Our previous\ngene profile analysis of the EOC peritoneum suggested\nthat CXCL8 (IL-8) has a central role in these inflammatory\ncell pathways [9,12]. Analysis of the mined data for both\nsPLA2 (group 2a) and cPLA2 (group 4a) revealed signifi-\ncant differences in transcript levels, being higher in EOC\ntumor (n = 8) or peritoneum (n = 10) than in benign per-\nitoneum (n = 5). These differences were statistically signif-\nicant for cPLA2 and sPLA2 respectively in EOC vs benign\nperitoneum (P = 0.004) and P = 0.02 and for both cPLA2\nand sPLA2 in EOC tumor vs benign peritoneum (P =\n0.005 for both). The P values were based on a nonpara-\nmetric rank-sum test for comparing samples with multiple\nendpoints [14].\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 5 of 11\n(page number not for citation purposes)\nPeritoneum biopsy specimens from women with benign\npelvic conditions contained fewer leukocytes (Fig. 2) than\ndid peritoneal specimens from patients with EOC, which\ndemonstrated more extensive leukocyte infiltration. Indi-\nrect immunoperoxidase staining with anti-CD68 or anti-\nCD3 monoclonal antibodies revealed that the leukocyte\ninfiltrates consisted of two main populations: MO/MA\n(CD68+) and T cells (CD3+). LCA was expressed by most\nperitoneal tissue leukocytes in both malignant and benign\nconditions. Other cell populations, including granulo-\ncytes, B cells, and NK cells, were rare (data not shown).\nCD68+ (MO/MA) and CD3+ (T-cell) infiltrates were\nexamined in tumor-free peritoneal tissues from 19 of the\n20 patients with EOC (one sample was unsuitable for\nassessment). The mean number of MO/MA per 0.08 mm2\nfield in the EOC peritoneum was 16.8 and that of T cells\nwas 11.2, as compared with 6.4 MO/MA and 2.6 T cells\nper field in benign peritoneum (Table 2). MO/MA were\nsubstantially more common than were T cells (Table 2) (P\n= 0.0002 by paired t-test). By contrast, the mean numbers\nof B cells (CD20+) and NK cells (CD56+) were 4.5 and 1.6\nper field in the EOC peritoneum respectively but were\nrarely detected in benign peritoneum.\nNumbers of CD68+ and CD3+ cells were also examined\nin paired samples where peritoneal tissues were available\nfrom each side of the pelvis. Paired t tests showed no sig-\nnificant differences between the two sides (Table 3). This\nbilateral presence of MO/MA and T-cell infiltrates in the\npelvic peritoneum suggests a spatially generalized distri-\nbution pattern rather than a site-specific effect. In the 5\nEOC cases in which paired superficial (submesothelial\nand deeper stroma) samples were available for compari-\nson, more CD3+ cells were found near the mesothelial\nsurface than in the deeper stroma (P = 0.020). CD68+\ncells were present in large numbers at both levels.\nWe next examined the MO/MA population, our primary\nfocus for this study, at the cellular proteomic level. The\nmonoclonal antibodies (mAbs) used included those that\nrecognize CD163, CCR1, CXCL8, VCAM1 and cPLA2.\nIn the present experiments, we examined the two main\nmononuclear cell populations, CD68+ and CD3+, by\nindirect immunofluorescence costaining using confocal\nmicroscopy, for characteristics that would help to identify\nfunctional subsets. Because CD68+ cells were the domi-\nnant population in the peritoneum, we reasoned that\nCD68+ cells (or some subset of those cells) could be a\nmajor source of the CXCL8 gene expression product in the\nperitoneum.\nPeritoneal biopsy specimens from 7 patients and ascites\ncytopreparations from 3 patients were tested with triple-\nantibody staining and evaluated by confocal microscopy.\nFigure 3 (Row 1) shows surface peritoneum with positive\nstaining for cytokeratin, CD31 (endothelial cells), and\nCD68. Marked infiltration of CD68+ cells is seen both\nbelow and within the surface mesothelium. This is com-\npared with the peritoneum from a patient with a benign\ncondition where only the keratin positive surface mes-\nothelium is shown along with some endothelial cells (Fig.\n3, Row 2). The proportions of CD68+ and CD3+ mono-\nnuclear leukocytes that coexpress products of certain\ngenes differentially overexpressed in the peritoneal tran-\nscriptome profile (Figure 1) are shown in Table 4. Figure\n3 and Table 4 show that CXCL8 (IL8), which appears to\nhave a central position in the peritoneal inflammatory\ntranscriptome, was expressed more often on CD68+ cells\nthan on the CD68+ CD163+ subset. CD163 has been\nidentified specifically on a subset of differentiated MO/\nMA, and might have a role in adaptive immunity [15]. To\nsummarize the important observations, CCR1, the chem-\nSelected genes expressed at different levels in the peritoneum and stroma of patients with EOC vs in patients with benign pel-vic diseaseFigure 1\nSelected genes expressed at different levels in the peritoneum and stroma of patients with EOC vs in patients with benign pel-\nvic disease. The red bars indicate the malignant phenotype and the blue bars the benign controls. The significance level of each \ngene expression between benign and malignant phenotypes is presented as P(t2) values.\nGene s ymbol p value\nCD3 d elt a 0. 472\nCD3 gamma 0.068\nPL A2G2A 0. 002\nIL8 0.022\nCD163 0. 013\nVCAM1 0.015\nCCR1 0.043\nGene s ymbol p value\nCD3 d elt a 0. 472\nCD3 gamma 0.068\nPL A2G2A 0. 002\nIL8 0.022\nCD163 0. 013\nVCAM1 0.015\nCCR1 0.043\nGene s ymbol p value\nCD3 d elt a 0. 472\nCD3 gamma 0.068\nPL A2G2A 0. 002\nIL8 0.022\nCD163 0. 013\nVCAM1 0.015\nCCR1 0.043\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 6 of 11\n(page number not for citation purposes)\nokine receptor that binds to a number of different ligands\nproduced in the environment of EOC was expressed on\n60.3% (range 44–83%) of CD68+ cells and on only\n15.1% (range 0–53%) of CD3+ cells in the peritoneum.\nProportions of CCR1 expression were 80.7% and 4% on\nascitic CD68+ and CD3+ cells, respectively. The propor-\ntion of CD68+ cells or CD68+ CCR1+ cells that coex-\npressed CXCL8 was also higher than in the CD3+ or CD3+\nCCR1+ populations. These results suggest that peritoneal\nMO/MA might be an important source of CXCL8 in the\nperitoneal environment of EOC.\nWe found the proportion of peritoneal CD68 + cells that\ncoexpressed CD163 to vary from 19% to 78%; cells that\nwere CD68+CD163+CXCL8+ usually contributed less\nthan 10% of total CD68+ cells (Figure 3, rows 3&4 &\nTable 4). In the EOC peritoneum, though a higher propor-\ntion were present in ascites, CD68+ cells were more often\ndetected within the surface layer of the mesothelium than\nwere CD163+ cells, which seemed to be concentrated\nbelow the mesothelial surface. However, in benign condi-\ntions, CD163+ cells, though present in smaller numbers,\nseemed to be more broadly distributed under the mes-\nMO/MA & T-cell infiltration in peritoneumFigure 2\nMO/MA & T-cell infiltration in peritoneum. Left upper shows peritoneum of patient w/benign fibrothecoma with scant \nLCA+ leukocytes below the single layer of mesothelium. Remaining 5 panels show tumor-free peritoneum from a patient with \nEOC. Upper middle shows large number of LCA+ cells; upper right shows large number of CD68+ cells; lower left shows rel-\natively fewer CD3+ cells; lower middle shows negative isotype control; lower right shows H&E. Magnification---200×\nEOC Isotype ControlEOC CD3 \nEOC CD68EOC LCABenign LCA\nEOC H&E\nEOC Isotype ControlEOC CD3 \nEOC CD68EOC LCABenign LCA\nEOC Isotype ControlEOC CD3 \nEOC CD68EOC LCABenign LCA\nEOC H&E\nEOC H&E\nTable 2: Median numbers of immune cells expressing CD3, CD68, and LCA in peritoneal tissue from patients with EOC or benign \novarian tumors\nCD3 (T cells) CD68 (MO/MA) CD45 (LCA)\nLeft Right Average Left Right Average Left Right Average\nEOC 7.4 9.5 8.7 14.7 15.3 15.3 17.4 18.2 18.1\nBenign 1.7 2.0 1.7 7.2 4.1 5.3 3.7 4.7 4.1\nP-value 0.012 0.016 0.002 0.038 0 .002 <0.001 0.003 0.001 <0.001\nValues are expressed as median absolute numbers of cells per 0.08-mm2 field.\nMO/MA, monocytes/macrophages; LCA, leukocyte common antigen.\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 7 of 11\n(page number not for citation purposes)\nothelium. The antibody to CD68 used here recognizes a\nlysozyme marker that can sometimes be coexpressed by\nkeratin-positive epithelial cells [16]. Some large lymphob-\nlastoid T-cells may also coexpress CD68, as suggested by\nthe presence of large ascitic mononuclear cells showing\nsurface staining for CD3 and cytoplasmic staining for the\nCD68 antigen. CD3 costaining with CD68 + or with\nCD163+ was rarely observed in the peritoneum, support-\ning the specificity of the antibody staining for T cells, total\nMO/MA, and the CD163+ MO/MA subset. CXCL8 was\nexpressed on the surface epithelium in both malignant\nand benign conditions (data not shown), suggesting that\nCXCL8, even at low levels, may have a functional role in\nthe absence of cancer.\nVCAM1, an important adhesion molecule, was also\nexpressed primarily by CD68+ cells (53%) and by CD31 +\nendothelial cells but only infrequently by CD3 + cells\n(3.6%) (Table 4). Finally, CD68 + cells and the CD163+\nsubset in both peritoneal and ascitic cells also expressed\nsubstantial amounts of pcPLA2 (Figure 4). However, the\npattern of pcPLA2 expression both in the peritoneum and\nin ascitic cells seemed to differ between the total popula-\ntion of CD68 + cells and the CD163 + cells; CD68+ cells\nshowed both cytoplasmic and nuclear staining, and\nCD163+ cells showed costaining of pcPLA2 only in the\nnucleus.\nConclusion\nCollectively, our results represent the first steps in show-\ning that inflammatory cells have a spatially generalized\ndistribution pattern in the pelvic peritoneum of EOC and\nthat the inflammatory cell subsets are both quantitatively\nand qualitatively different from the patterns typical of\nbenign pelvic disease. These findings complement those\nof our previous study of the EOC peritoneal transcriptome\n[9] and could suggest a common biologic effect. The\ninflammatory cell infiltrates in cancer could contribute to\nantitumor effects or, conversely, promote invasion and\nmetastasis. Our previous studies [17,18] suggest that\nascitic MO/MA, representing a substantial proportion of\nthe intraperitoneal inflammatory cell environment,\ninclude cells that exhibit defective Fc γ R [18]mediated\nactivity or mediate T cell suppressor functions[17]. Here\nwe showed that tissue from nontumor-involved perito-\nneum in patients with EOC exhibited substantial leuko-\ncyte infiltrates in comparison with peritoneal tissues in\npatients with benign pelvic disease and that the infiltrate\nconsisted mainly of MO/MA and, to a lesser extent, T cells.\nOther cells (NK [CD56+] and B cells [CD20+]) were\nfound, but in much lower numbers. These results lead us\nto speculate that MO/MA and T-cell infiltrates in the peri-\ntoneum were responding to a general migration stimulat-\ning effect; given their proximity to the peritoneal cavity,\nthe large numbers of MO/MA and T cells found in ascitic\nfluid could well have originated from cells that had\nmigrated into the submesothelial stroma from an exten-\nsive network of small capillary vessels, facilitated by\nexpression of adhesion molecules in the capillary\nendothelium. These views are consistent with the work of\nAlberto Mantovani [19,20] who has demonstrated the\neffect of tumor cell products on the \"polarization\" of MA.\nIn our studies on the peritoneum, we found substantial\nnumbers of CD68+ cells, and CD68+ CD163+ cells, to be\nconcentrated near the mesothelium. The CD68+ CD163 -\npopulation in particular appeared more likely to coex-\npress CXCL8, a proangiogenic chemokine that can influ-\nence the migration of different leukocyte populations. The\npresence of CD68 cells within the mesothelial cell layer\nalso suggests that these cells are in transit to the peritoneal\ncavity compartment or ascitic fluid.\nA number of chemokines might contribute to the migra-\ntion and activation of leukocytic as well as other cells in\nthe EOC environment. Migration effect is dependent\nlargely on the expression of complementary receptors of\nascites for a number of CC or CXC chemokines (named\nfor the arrangement of their first two cysteine residues). As\nTable 3: The comparison of immune cells with positive CD3, CD68, and LCA markers between left and right sides in patients with \nEOC.\nMarker and Location No. of Samp les Average Difference (Left – \nRight)\nP Value\nCD3\nPeritoneum 15 Median -0.33 0.89\nStroma 5 Median -2.40 0.42\nCD68\nPeritoneum 15 Median 0.57 0.84\nStroma 5 Median 0.10 0.97\nLCA\nPeritoneum 15 Median 1.27 0.77\nStroma 5 Median -4.90 0.29\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 8 of 11\n(page number not for citation purposes)\nwe have shown here and elsewhere [12,21], CXCL8\nappears to have a prominent role in the peritoneal and\nascitic CD68+ population and specifically in CCR1+ cells.\nAt least nine CC chemokines, many of them associated\nwith EOC [21,22], can serve as ligands for CCR1. Here we\nfound CCR1, also highly expressed in the peritoneal\nmicroarray profile, on substantial numbers of peritoneal\nMO/MA and on some T cells, suggesting that CCR1, could\nplay an important role in migration of certain cell popu-\nlations that express this receptor.\nCXCL8 can be induced by various cytokines, including IL-\n1, TNFα , IL-3, IL-13, and IL-7, most of which are produced\nin EOC patients and can be induced H 2O2 and hypoxia.\nCXCL8 binds to CXCR1 or CXCR2, either of which can be\nexpressed on resting T cells but are not usually on mono-\ncytes. We found CXCL8 to be produced on a large propor-\ntion of MO/MA and, more variably, on CD3+ cells.\nMoreover, only a very low proportion (< 10%) of the\nCD68+CD163+ subset produced CXCL8. CD163 has\nbeen linked with IL-10 release in atheromatous disease\n[23], though in the pig, it has been associated with adap-\ntive immunity [15]. The functional role of CD163+ MO/\nMA in EOC is yet to be determined.\nTriple immunofluorescence costaining of frozen right peritoneal tissues were stained with CD68 (red), CD31 (blue), and kera-tin (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 cellsFigure 3\nTriple immunofluorescence costaining of frozen right peritoneal tissues were stained with CD68 (red), CD31 (blue), and kera-\ntin (CK) (green) antibodies (Rows 1 & 2) Row 1, peritoneal cells from a patient with EOC (ID 266 m) appear yellow from the \ncolocalization of CD68 (red) and CK (green) on some surface mesothelial cells. CD31 staining (blue) indicates endothelial cells \njust under the mesothelium. Row 2, peritoneal cells from a patient with benign cystic teratoma of the ovary (ID 283b) show \nprominent staining for keratin in the single cell mesothelial layer but no staining for CD68 staining (red) and positive staining \nfor endothelial cells (blue). Rows 3 and 4, peritoneal cells from patient ID#235 showed colocalization of CD68 (blue) and \nCD163 (green) appearing cyan color; CD68 (blue) and CXCL8 (red) costaining showed magenta effect and no color changed in \nCD163+ cells (green). Images were analyzed by confocal laser scanning microscopy (magnification 400×). H&E stained sections \nare shown for comparison.\nCD68/CK CD68/CD31 CD31/CK CD68/CD31/CK\nDIC \n(Overlay)\nID#283b\nID#266m\nH&E\nID#235\nCD68 CD163 IL8\nCD68/CD163 CD163/IL8 CD68/IL8 CD68/CD163/IL8\nH&E\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 9 of 11\n(page number not for citation purposes)\nElevated levels of the pro-angiogenic chemokine CXCL8\n(IL8) have been detected in a variety of tumors, including\nsolid EOC [24] and EOC ascites fluid [25] and may pro-\nmote tumor growth. Low levels have also been detected in\nserum of certain normal subjects, and we have observed\nthat the single layer of mesothelium in benign pelvic dis-\nease may be positive for this cytokine (data not shown).\nUnpublished data from the Human Cancer Immunology\nResearch Core Facility of M. D. Anderson Cancer Center,\nmoreover, indicate that 8 of 40 normal donors (all\nfemales) had IL-8 levels higher than the lowest standard\nof 9 pg/ml (Dr. James Reuben, personal communication).\nThese findings suggest that lower levels of CXCL8 might\nhave a physiologic role.\nCells that produce CXCL8 in the peritoneum of patients\nwith EOC might be expected to contribute to elevated lev-\nels of CXCL8 in the peritoneal, ascitic, and blood com-\npartments of such patients. CXCL8 is likely to have an\nimportant role in the development or spread of EOC; it,\nalong with vascular endothelial growth factor, has been\nlinked with unfavorable prognosis in EOC [26]. The TNF-\nrelated apoptosis-inducing ligand (TRAIL) can trigger\napoptosis in many malignant cells [27], but CXCL8 has\nbeen shown to block TRAIL-induced cell death by convert-\ning a TRAIL-sensitive ovarian cancer cell line (OVCAR3)\ninto a TRAIL-resistant one. CXCL8 may also regulate the\nexpression of a member of the mitogen-activated protein\nkinase superfamily, p38 γ [28] and with VEGF contribute\nto increased endothelial capillary functions. Lysophos-\nphatidic acid (LPA), a phospholipid produced from\nmalignant ovarian epithelium, can enhance the expres-\nsion of CXCL8 by tumor cells and stimulate EOC cell inva-\nsion by enhancing membrane type-1 (MT1) matrix\nmetalloproteinase (MMP) mediated activation of MMP2\n[29]. Interestingly, cPLA2, an activated enzyme involved\nin liberating arachidonic acid from cell membranes and\ndependent on MAPK-induced phosphorylation [30] was\nhighly expressed in CD68+ cells and the CD68+CD163+\nsubset. p42/44 and p38 MAPK activation is required prior\nto translocation to the nucleus [31]. Activation of cPLA2\nand other phosphorylates by cytokines in the environ-\nment of EOC may contribute to CXCL8 production. Ara-\nchidonic acid is the precursor of fatty acid derivatives,\nincluding LPA, leukotrienes, prostaglandins, thrombox-\nanes, and other important components of the eicosanoid\npathways. LPA activates several biological responses\nthrough its binding and activation of G-protein-coupled\nreceptors, and has been detected at elevated levels in the\nascites and serum of patients with EOC [32].\nWe might speculate that chemokines and certain\ncytokines could be involved in recruiting MO/MA and cer-\ntain T cells into the submesothelial stroma of the perito-\nneum, where such cells could contribute to tissue\nreorganization, tumor cell invasion, angiogenesis, capil-\nlary leakage, and the production of ascites.\nThe cytokines most often detected in serum and ascites of\npatients with EOC include TNF α , IL-10, IL-6, CSF1\nTable 4: Proportions and mean proportions across samples for CD68+ and CD3+ mononuclear leukocytes expressing CCR1, IL-8, and \nVCAM1 by confocal microscopy.\nANTIBODIES PERITO NEAL SPECIM ENS ASCITES\nTS-266 TS-265 TS-235 TS-236 TS-267 TS-242 TS-256 M eans ASC290 ASC288 ASC278 Means\nCD68+/CCR1+ 65 51 45 44 76 58 83 60.3 87 77 78 80.7\nC D 6 8 + / I L 8 + 6 13 53 83 64 56 67 4 5 0 . 75 5 5 2 4 2 4 9 . 7\nCD68+/CCR1+IL8+ 66 38 28 30 46 42 37 41 45 35 48 42.7\nC D 3 + / C C R 1 + 5 3 1 9 1 8 6208 1 5 . 1 0 1 0 24\nCD3+/IL8+ 80 45 19 34 7 4 8 28.1 0 10 2 4\nCD3+/CCR1+IL8+ 47 24 5 11 0 0 0 12.4 0 10 2 4\nCD68+/CD163+ 19 38 31 43 67 39 78 45 30 36 91 52.3\nCD163+/IL8+ 22 7 0 20 11 45 22 18.1 60 65 26 50.3\nCD68+/CD163+IL8+ 5 3 0 9 4 22 19 8.9 15 29 32 25.3\nCD68+/VCAM1+ 59 94 68 60 55 4 29 52.7 79 81 26 62\nC D 6 8 + / C D 3 + 1012063 1 . 9 1 93 31 5 2 2 . 3\nC D 3 + / V C A M 1 + 405400 1 2 3 . 6 3 86 93 1 4 6\nC D 6 8 + / C D 3 + V C A M 1 + 1010002 0 . 6 1 63 1 9 1 8 . 7\nCD3/CD68 Ratio 1 to 2 1 to 11 1 to 4 1 to 4 1 to 2 1 to 2 1 to 8 1 to 2 1 to 2 1 to 2\nDisease Stage III III III III III II II III III III\nHistology C E S/E S/E S S/E E S S S\nS = serous; E = endometriod; C = clear cell\n\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 10 of 11\n(page number not for citation purposes)\n(MCSF), IL-1, and TGFβ isotypes, all of which can be pro-\nduced by activated MA [17,33-37] or by the tumor cells\n[10,38]. TNFα  and IL-1α  enhance the expression of adhe-\nsion molecules on endothelial cells, thereby contributing\nto leukocyte attachment and migration. In our study,\nVCAM1 was coexpressed on both CD31+ endothelial cells\nand on MO/MA but not on most CD3+ cells, suggesting\nthat the CD68+ cells and CD31+ endothelial cells in par-\nticular might be under the influence of VCAM1-sensitive\ncytokines released into the peritoneal microenvironment.\nVCAM1 is upregulated on cytokine stimulated endothe-\nlium. Such a release could be an important early step in\nthe migration behavior of MO/MA into the peritoneum\nand ascitic fluid. The presence of endothelial cells\n(CD31+) in proximity to and the CD68+ cells either inter-\nspersed in or just under the mesothelium, suggest that\nthere is a close spatial relationship of peritoneal MO/MA\nto ascitic MO/MA.\nIn summary, this study represents the first description of\nthe inflammatory cell response in the peritoneum of\npatients with EOC. Our findings here support and com-\nplement our previous gene profiling study of the perito-\nneum [9]. We conclude that peritoneal MA that are\nseparated spatially from tumor cells share several similar\nphenotypic characteristics and express activated gene\nproducts that could have important roles in tumor growth\nand metastases.\nReferences\n1. Rubin SC, Randall TC, Armstr ong KA, Chi DS, Hoskins WJ: Ten-\nyear follow-up of ovarian canc er patients af ter second-look\nlaparotomy with ne gative findings.   Obstet Gynecol  1999,\n93:21-24.\n2. Dedrick RL, Flessner MF: Pharmacokinetic problems in perito-\nneal drug administration: tissue penetration and surface\nexposure.  J Natl Cancer Inst 1997, 89:480-487.\n3 . C a r t e r  D ,  T r u e  L ,  O t i s  C :  Serous Membranes.   In Histology for\nPathologists, 2nd Edition  Edited by: Sternberg S.  Philadelphia, Lippin-\ncott-Raven; 1997:223-239. \n4. Battifora H, McCaughey W: Tumors of the serosal membranes.\nIn Atlas of tumor pathology, Third series  Volume 1. Edited by: Pathology\nUARE. Bethesda, Armed Forces Institute of Pathology; 1995. \n5. Freedman RS, Deavers M, Liu J, Wang E: Peritoneal inflammation\n- A microenvironment for Epithelial Ovarian Cancer (EOC).\nJ Transl Med 2004, 2:23.\n6. Wang E, Marincola FM: Amplification of small quantities of\nmRNA for transcript analysis.  In DNA arrays—amolecular cloning\nmanual 1st ed Edited by: Bowtell D and J S. Cold Spring Harbor Lab-\noratory, Cold Spring Harbor (NY); 2002:204-213. \nStained 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)Figure 4\nStained cytospin preps of mononuclear leukocytes isolated from ascitic fluid on F:H density cushion (patient ID 290; top row) \nor from frozen tissue (patient ID 235; bottom row) were fixed with 4% paraformaldehyde and double-stained with phospho-\ncPLA2 (Ser505) antibody (red) and CD68 (green) or CD163 (green). Indirect immunofluorescence showed pcPLA2 in both the \nnucleus and cytoplasm, and results were similar in the cells from ascites and those from tissue. Cells costained for CD68 and \ncPLA2 showed colocalization of both markers in the cytoplasm and in the nucleus (yellow). However, in CD163+ cells, cPLA2 \nwas seen only in relation to the nucleus.\ncPLA2 CD68/cPLA2 CD163/cPLA2\nTS-ID#235 ASC-ID#290\n\nPublish with BioMed Central   and  every \nscientist can read your work free of charge\n\"BioMed Central will be the most significant development for \ndisseminating the results of biomedical research in our lifetime.\"\nSir Paul Nurse, Cancer Research UK\nYour research papers will be:\navailable free of charge to the entire biomedical community\npeer reviewed and published immediately upon acceptance\ncited in PubMed and archived on PubMed Central \nyours — you keep the copyright\nSubmit your manuscript here:\nhttp://www.biomedcentral.com/info/publishing_adv.asp\nBioMedcentral\nJournal of Translational Medicine 2006, 4:30 http://www.translational-me dicine.com/content/4/1/30\nPage 11 of 11\n(page number not for citation purposes)\n7. Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM: High-fidel-\nity mRNA amplification for gene profiling.  Nat Biotechnol 2000,\n18:457-459.\n8. Wang E: RNA amplification for successful gene profiling anal-\nysis.  J Transl Med 2005, 3:.\n9. Wang E, Ngalame Y, Panelli MC , Nguyen-Jackson  H, Deavers M,\nMueller P, Hu W, Savary CA, Ko bayashi R, Freedman RS, Marincola\nFM: Peritoneal and Subperitoneal Stroma May Facilitate\nRegional Spread of Ovarian Cancer.   Clin Cancer Res  2005,\n11:113-122.\n10. Gordinier ME, Zhang HZ, Patenia R, Levy LB, Atkinson EN, Nash MA,\nKatz RL, Platsoucas CD, Freedman RS: Quantitative analysis of\ntransforming growth factor-beta (TGF-beta 1 and TGF-\nbeta2) on ovarian carcinoma.   Clinical Cancer Research  1999,\n5:2498-2505.\n11. Freedman RS, Lenzi R, Kudelka AP, Rosenblum M, Platsoucas CD:\nIntraperitoneal immunotherapy of peritoneal carcinomato-\nsis.  Cytokines, Cellular and Molecular Therapy 1998, 4:121-140.\n12. Wang X, Wang E, Kavanagh JJ, Freedman RS: Ovarian cancer, the\ncoagulation pathway, and inflammation.   J Transl Med  2005,\n3:25.\n13. Ross DT, Scherf U, Eisen MB, Perou CM, Rees C, Spellman P, Iyer V,\nJ e f f e r y  S S ,  V a n  d e  R i j n  M ,  W a l tham M, Pergamenschikov A, Lee J,\nLashkari D, Shalon D, Myers TG, Weinstein JN, Botstein D, Brown\nPO: Systematic variation in ge ne expression patterns in\nhuman cancer cell lines.  Nature Genetics 2000, 24:227-235.\n14. O'Brien PC: Procedures for comparing samples wtih multiple\nendpoints.  Biometrics 1984, 40:1079.\n15. Chamorro S, Revilla C, Alvarez B,  Alonso F, Esquerra A, Dominguez\nJ: Phenotypic and functional hete rogeneity of porcine blood\nmonocytes and its relation with maturation.  Immunology 2005,\n114:63-71.\n16. Ordonez NG, Freedman RS, Herlyn M: Lewis and related tumor-\nassociated determinants on ovarian carcinoma.  Gynecol Oncol\n1987, 26:1-10.\n17. Loercher AE, Nash MA, Kavanagh JJ, Platsoucas CD, Freedman RS:\nIdentification of an IL-1 0-producing HLA-DR-negative\nmonocyte subset in the maligna nt ascites of patients with\novarian carcinoma that inhibits cytokine protein expression\nand proliferation of autologous T cells.   J Immunol  1999,\n163:6251-6260.\n18. Gordon IO, Freedman RS: Defective antitumor function of\nmonocyte-derived macrophages from epithelial ovarian can-\ncer patients.  Clin Cancer Res 2006, In press:.\n19. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A: Macrophage\npolarization: tumor-associated macrophages as a paradigm\nfor polarized M2 mononuclear phagocytes.   Trends Immunol\n2002, 23:549-555.\n20. Orre M, Rogers PA: Macrophages and microvessel density in\ntumors of the ovary.  Gynecol Oncol 1999, 73:47-50.\n21. Negus RP, Stamp GW, Relf MG, Bu rke F, Malik ST, Bernasconi S,\nAllavena P, Sozzani S, Mantovani A, Balkwill FR: The detection and\nlocalization of monocyte chem oattractant protein-1 (MCP-\n1) in human ovarian cancer.  J Clin Invest 1995, 95:2391-2396.\n22. Schutyser E, Struyf S, Proost P, Opdenakker G, Laureys G, Verhasselt\nB, Peperstraete L, Van de Putte I, Saccani A, Allavena P, Mantovani A,\nVan Damme J: Identification of biolog ically active chemokine\nisoforms from ascitic fluid and elevated levels of CCL18/pul-\nmonary and activation-regulated chemokine in ovarian car-\ncinoma.  J Biol Chem 2002, 277:24584-24593.\n23. Philippidis P, Mason JC, Evans BJ , Nadra I, Taylor KM, Haskard DO,\nLandis RC: Hemoglobin scavenger recepter CD163 mediates\ninterleukin-10 release and heme oxygenase-1 synthesis: anti-\ninflammatory monocyte-macrophage responses in vitro, in\nresolving skin blisters in vi vo, and after cardiopulmonary\nbypass surgery.  Circulation Research 2004, 94:119-126.\n24. Ivarsson K, Ekerydh A, Fyhr IM, Janson PO, Brannstrom M: Upregu-\nlation of interleukin-8 and po larized epithelial expression of\ninterleukin-8 receptor A in ovarian carcinomas.   Acta Obstet\nGynecol Scand 2000, 79:777-784.\n25. Gawrychowski K, Skopinska-Rozewska E, Barcz E, Sommer E, Sza-\nniawska B, Roszkowska-Purska K, Janik P, Zielinski J: Angiogenic\nactivity and interl eukin-8 content of human ovarian cancer\nascites.  Eur J Gynaecol Oncol 1998, 19:262-264.\n26. Kassim SK, El-Salahy EM, Fayed ST , Helal SA, Helal T, Azzam Eel D,\nKhalifa A: Vascular endothelial growth factor and interleukin-\n8 are associated with poor pr ognosis in epithelial ovarian\ncancer patients.  Clin Biochem 2004, 37:363-369.\n27. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK,\nSutherland GR, Smith TD, Rauch C, Smith CA, et al.: Identification\nand characterization of a ne w member of the TNF family\nthat induces apoptosis.  Immunity 1995, 3:673-682.\n28. Abdollahi T, Robertson NM, Abdollahi A, Litwack G: Identification\nof interleukin 8 as an inhibi tor of tumor necrosis factor-\nrelated apoptosis-in ducing ligand-induced  apoptosis in the\novarian carcinoma cell line OVCAR3.   Cancer Res  2003,\n63:4521-4526.\n29. So J, Navari J, Wang FQ, Fishman DA: Lysophosphatidic acid\nenhances epithelial ovarian carcinoma invasion through the\nincreased expression of interleukin-8.   Gynecol Oncol  2004,\n95:314-322.\n30. Boonstra J, Verkleij AJ: Regulation of enzyme activity in vivo is\ndetermined by  its cellular localization.  Advances in Enzyme Reg-\nulation 2004, 44:61-73.\n31. Grewal S, Morrison EE, Ponnambalam S, Walker JH: Nuclear local-\nization of cytosolic phospholipase A2-alpha in the EA.hy.926\nhuman endothelial cell line is  proliferation dependent and\nmodulated by phosphorylation.  J of Cell Science 2002, 115:.\n32. Xu Y, Shen Z, Wiper DW, Wu M, Morton RE, Elson P, Kennedy AW,\nBelinson J, Markman M, Casey G: Lysophosphatidic acid as a\npotential biomarker for ovarian and other gynecologic can-\ncers.  Jama 1998, 280:719-723.\n33. Freedman RS, Edwards CL, Kavan agh JJ, Kudelka AP, Katz RL, Car-\nrasco CH, Atkinson EH, Scott W, Tomasovic B, Platsoucas CD:\nIntraperitoneal adoptive immu notherapy of ovarian carci-\nnoma with tumor infiltrating lymphocytes, A pilot trial.   J\nImmunother 1994, 16:198-210.\n34. Nash MA, Lenzi R, Edwards CL, Ka vanagh JJ, Kudelka AP, Verschrae-\ngen CF, Platsoucas CD, Freedman RS: Differential expression of\ncytokine transcripts in huma n epithelial ovarian carcinoma\nby solid tumor specimens, pe ritoneal exudate cells contain-\ning tumor, TIL-derived T-cell lines and established tumor\ncell lines.  Clin Exp Immunol 1998, 112:172-180.\n35. Moradi MM, Carson LF, Weinberg  JB, Haney AF, Twiggs LB, Ram-\nakrishnan S: Serum and ascitic fluid le vels of interleukin-1,\ninterleukin-6, and tumor necros is factor-alpha in patients\nwith ovarian epithelial cancer.  Cancer 1993, 72:2433-2440.\n36. Burke F, Relf M, Negus RP, Balkwill FR: A cytokine profile of nor-\nmal and malignant ovary.  Cytokine 1996, 8:578-585.\n37. Berek JS, Chung C, Kaldi K, Watson JM, Knox RM, Martinez-Maza O:\nSerum interleukin-6 levels correlate with disease status in\npatients with epithe lial ovarian cancer.   Am J Obstet Gynecol\n1991, 164:1038-1042.\n38. Nash MA, Ferrandina G, Gordinie r ME, Loercher AE, Freedman RS:\nThe role of cytokines in bo th the normal and malignant\novary.  Endocrine-Related Cancer 1999, 6:93-107.","source_license":"CC0","license_restricted":false}