Characteristics and Growth Patterns of Human Peritoneal Mesothelial Cells: Comparison Between Advanced Epithelial Ovarian Cancer and Non-Ovarian Cancer Sources

Abstract

Objective: To compare the characteristics and growth patterns of human peritoneal mesothelial cells (HPMCs) from advanced epithelial ovarian cancer (EOC) patients with those from non-EOC patients.

Methods: Peritoneal and omental biopsies were obtained from treatment-naïve patients. Formalin-fixed biopsies and cytologic touch preparations were studied immunochemically. HPMCs were isolated from tissue biopsies or malignant ascites and cultured with or without growth factors. Cell growth was determined by the MTT assay. Cultured HPMCs were further characterised by flow cytometry analysis (FACS).

Results: Peritoneal biopsies showed a continuous flat mesothelial cell layer in non-EOC patients, whereas in advanced EOC, the mesothelium was a discontinuous layer of rounded cells. In all peritoneal biopsies, the mesothelium expressed cytokeratin 8/18, vimentin, and the mesothelioma cell antigen but not E-cadherin. In touch preparations, expression of the putative fibroblast antigen ranged from negative to weakly positive. HPMC from non-EOC cases grew slowly in vitro except when exposed to L-cysteine (L-cys 30 μg/mL) during the initial 24 hours of culture. Conversely, cells from EOC sources grew more rapidly, especially when exposed to both epidermal growth factor (EGF 10 ng/mL) and hydrocortisone (HC 400 ng/mL). HPMC coexpressed cytokeratin 8/18 and vimentin in vitro, but the expression of the putative fibroblast antigen increased during primary culture, whereas that of the mesothelioma cell antigen decreased in successive passages. Furthermore, in FACS, cultured HPMC did not express CD14, CD16, or CD34.

Conclusion: In peritoneal biopsies from non-EOC and EOC patients, HPMCs showed different morphology but similar immunostaining characteristics, whereas cultured cells from different sources were similar in both morphology and phenotype. L-cysteine enhanced the growth of non-EOC but not of EOC-derived HPMCs, which had a maximal response to EGF and HC. The growth advantage of HPMCs from EOC in vitro suggests these cells are in a primed or activated state.

Keywords

Ovarian cancer peritoneal mesothelium mesothelial cells

Get full access to this article

View all access options for this article.

References

1. Shostak A , Chakrabar E , Hirszel P , Maher JF . Effects of histamine and its receptor antagonists on peritoneal permeability. Kidney Int 1988;34:786-790.

2. Muijsken MA , Heezius HJCM , Verhoef J , Verbrugh HA . Role of mesothelial cells in peritoneal antibacterial defence. J Clin Pathol 1991;44:600-604.

3. Nakashio T , Narita T , Akiyama S , et al. Adhesion of human gastric and pancreatic cancer cells to peritoneal mesothelial cells is mediated by CD44 and β1 integrin. Int J Oncol 1997 (a);10:183-188.

4. Nakashio T , Narita T , Akiyama S , et al. Peritoneal dissemination is inhibited by treatment with antibodies against CD44H, β1 integrin, and carcinostatic agents in NUGC-4 human gastric cancer cells. Int J Oncol 1997;10:355-362.

5. Wilson AP . Mesothelial cells stimulate the anchorage-independent growth of human ovarian tumor cells. Br J Cancer 1989;59:876-882.

6. Jones LMH , Gardner MJ , Catterall JB , Turner GA . Hyaluronic acid secreted by mesothelial cells: A natural barrier to ovarian cancer cell adhesion. Clin Exp Metastasis 1995;13:373-380.

7. Kishikawa T , Sakamoto M , Ino Y , Kubushiro K , Nozawa S , Hirohashi S . Two distinct patterns of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models. Invasion Metastasis 1995;15:11-21.

8. Strobel T , Swanson L , Cannistra SA . In vivo inhibition of CD44 limits intra-abdominal spread of a human ovarian cancer xenograft in nude mice: A novel role for CD44 in the process of peritoneal implantation. Cancer Res 1997;57:1228-1232.

9. Wu YJ , Parker LM , Binder NE , et al. The mesothelial kertains: A new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkertainizing epithelia. Cell 1982;31:693-703.

10.

10. Stylianou E , Jenner LA , Davies M , Coles GA , Williams JD . Isolation, culture and characterization of human peritoneal mesothelial cells. Kidney Int 1990;37:1563-1570.

11.

11. Bird SD , Legge M , Walker RJ . L-cysteine improves growth of human peritoneal mesothelial cells in vitro. Perit Dial Int 1996;16:599-606.

12.

12. Kimura A , Koga S , Kudoh H , Litsuka Y . Peritoneal mesothelial cell injury factors in rat cancerous ascites. Cancer Res 1985;45:4330-4333.

13.

13. Yashiro M , Chung YS , Inoue T , et al. Hepatocyte growth factor (HGF) produced by peritoneal fibroblasts may affect mesothelial cell morphology and promote peritoneal dissemination. Int J Cancer 1996;67:289-293.

14.

14. Hsu SM , Raine L , Fanger H . Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577-580.

15.

15. Fickling SA , Tooze JA , Whitley GSJ . Characterization of human umbilical vein endothelial cell lines produced by transfection with the early region of SV40. Exp Cell Res 1992;201:517-521.

16.

16. Mosmann T . Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytoxicity assays. J Immunol Methods 1983;65:55-63.

17.

17. Sheibani K , Esteban JM , Bailey A , Battifora H , Weiss LM . Immunopathologic and molecular studies as an aid to the diagnosis of malignant mesothelioma. Hum Pathol 1992;23:107-116.

18.

18. Larocca P , Rheinwald JG . Coexpression of simple epithelial keratins and vimentin by human mesothelium and mesothelioma in vivo and in culture. Cancer Res 1984;44:2991-2999.

19.

19. Van-Niekerk CC , Jap PHK , Thomas CMG , Smeets DFCM , Ramaekers FCS , Poels LG . Marker profile of mesothelial cells versus ovarian carcinoma cells. Int J Cancer 1989;43:1065-1071.

20.

20. Miettinen M , Kovatich AJ . HBME-1: A monoclonal antibody useful in the differential diagnosis of mesothelioma, adenocarcinoma, and soft-tissue and bone tumors. Appl Immunohistochem 1995;3:115-122.

21.

21. Ascoli V , Carnovale-Scalzo C , Taccogna S , Nardi F . Utility of HBME-1 immunostaining in serous effusions. Cytopathology 1997;8:328-335.

22.

22. Schofield K , D'Aquila T , Rimm DL . The cell adhesion molecule, E-cadherin, distinguishes mesothelial cells from carcinoma cells in fluids. Cancer Cytopathology 1997;81:293-298.

23.

23. Akedo H , Shinkai K , Mukai M , et al. Interaction of rat ascites hepatoma cells with cultured mesothelial cells layers: A model for tumor invasion. Cancer Res 1986;46:2416-2422.

24.

24. Verger C , Luger A , Moore HL , Nolph KD . Acute changes in peritoneal morphology and transport properties with infections, peritonitis and mechanical injury. Kidney Int 1983;23:823-831.

25.

25. Connell ND , Rheinwald JG . Regulation of the cytoskeleton in mesothelial cells: Reversible loss of keratin and increase in vimentin during rapid growth in culture. Cell 1983;34:245-253.

26.

26. Moll R , Franke W , Schiller D . The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell 1982;31:11-24.

27.

27. Potzsch B , Grulich-Henn J , Rossing R , Wille D , Muller-Berghaus G . Identification of endothelial and mesothelial cells in human omental tissue and in omentum-derived cultured cells by specific cell markers. Lab Invest 1990;6:841-852.

28.

28. Jahn L , Fouquet B , Robe K , Franke W . Cytokeratins in certain endothelial and smooth muscle cells of two taxonomically distant vertebrate species, Xenopus laevis and man. Differentiation 1987;36:234-254.

29.

29. Saalbach A , Anderegg U , Bruns M , Schnabel E , Herrmann K , Haustein UF . Novel fibroblast-specific monoclonal antibodies: Properties and specificities. J Invest Dermatol 1996;106:1314-1319.

30.

30. Thomas E , Al-Baker E , Dropcova S , et al. Different kinetics of senescence in human fibroblasts and peritoneal mesothelial cells. Exp Cell Res 1997;236:355-358.