Abstract
Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18+/PSA− HT29 colon carcinoma cells. CK18+ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hypertrophy. In conclusion, the approach presented appears to be a reliable method to phenotype individual prostatic carcinoma cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Blatt WF, Robinson SM (1968) Membrane ultrafiltration: the diafiltration technique and its application to microsolute exchange and binding phenomena. Anal Biochem 26:151–173
Bonnard C, Papermaster DS, Kraehenbuhl J-P (1984) The streptavidin-biotin bridge technique: application in light and electron microscope immunocytochemistry. In: Polak JM, Varndell IM (eds) Immunolabelling for electron microscopy. Elsevier, Amsterdam, pp 95–111
Bosch FX, Leube RE, Achtstätter T, Moll R, Franke WW (1988) Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ. J Cell Biol 106:1635–1648
Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32:219–229
Cote RJ, Rosen PP, Lesser ML, Old LJ, Osborne MP (1991) Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases. J Clin Oncol 9:1749–1756
Danscher G (1981) Localization of gold in biological tissue. A photochemical method for light and electron microscopy. Histochemistry 71:81–88
Debus E, Weber K, Osborn M (1982) Monoclonal cytokeratin antibodies that distinguish simple from stratified squamous epithelia: characterization on human tissues. EMBO J 1:1641–1647
Debus E, Moll R, Franke WW, Weber K, Osborn M (1984) Immunohistochemical distinction of human carcinomas by cytokeratin typing with monoclonal antibodies. Am J Pathol 114:121–130
De Valck V, Renmans W, Segers E, Leunissen J, De Waele M (1991) Light microscopical detection of leucocyte cell surface antigens with a one-nanometer gold probe. Histochemistry 95:483–490
Donn F, Bruns T, von Meyerinck L, Schulz M, Becker W-M, Becker H, Klosterhalfen H (1989) Monoclonal antibody that defines the prostate specific antigen. Prostate 14:237–249
Franke WW, Moll R (1987) Cytoskeletal components of lymphoid organs. Synthesis of cytokeratin 8 and 18 and desmin in subpopulation of extrafollicular reticulum cells of human lymph nodes, tonsil and spleen. Differentiation 36:145–163
Gallee MPW, Vroonhoven CCJ van, Korput HAGM van der, Kwast van der TH, Kate FJW ten, Romijn JC, Trapman J (1986) Characterization of monoclonal antibodies raised against the prostatic cell line PC-82. Prostate 9:33–45
Gerdes J, Schwab U, Lemke H, Stein H (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31:13–20
Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA, Murphy GP (1983) LNCaP model of human prostatic carcinoma. Cancer Res 43:1809–1818
Huang WM, Gibson SJ, Facer P, Gu J, Polak JM (1983) Improved section adhesion for immunocytochemistry using high molecular weight polymers of l-lysine as a slide coating. Histochemistry 77:275–279
Lin CT, Liu JW, Song GX, Wu JY, Lam KW, Yam LT, Li CY (1986) Immuno-ultrastructural demonstration of prostatic acid phosphatase isoenzyme 2 in prostatic carcinoma. J Urol 136:173–180
Lindemann F, Schlimok G, Dirschedl P, Witte J, Riethmüller G (1992) Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients. Lancet 340:685–689
Loos JA, Roos D (1974) Ficoll-Isopaque gradients for the determination of density distributions of human blood lymphocytes and other reticulo-endothelial cells. Exp Cell Res 86:333–341
Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982) The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24
Moll R, Achtstätter T, Becht E, Balcarova-Ständer J, Ittensohn M, Franke WW (1988) Cytokeratins in normal and malignant transitional epithelium. Am J Pathol 132:123–144
Nadji M, Tabei SZ, Castro A, Chu TM, Murphy GP, Wang MC, Morales AR (1981) Prostatic-specific antigen: an immunohistologic marker for prostatic neoplasms. Cancer 48:1229–1232
Pantel K, Schlimok G, Kutter D, Schaller G, Genz T, Wiebecke B, Backmann R, Funke I, Riethmüller G (1991) Frequent down-regulation of major histocompatibility class I antigen expression on individual micrometastatic carcinoma cells. Cancer Res 51:4712–4715
Roth J (1982) Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections. J Histochem Cytochem 30:691–696
Schlimok G, Funke I, Holzmann B, Göttlinger G, Schmidt G, Häuser H, Swierkot S, Warnecke HH, Schneider B, Koprowski H, Riethmüller G (1987) Micrometastatic cancer cells in bone marrow: In vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies. Proc Natl Acad Sci USA 84:8672–8676
Schlimok G, Funke I, Bock B, Schweiberer B, Witte J, Riethmüller G (1990) Epithelial tumor cells in bone marrow of patients with colorectal cancer: Immunocytochemical detection, phenotypic characterization, and prognostic significance. J Clin Oncol 8:831–837
Schlimok G, Funke I, Pantel K, Strobel F, Lindemann F, Witte J, Riethmüller G (1991) Micrometastatic tumour cells in bone marrow of patients with gastric cancer: methodological aspects of detection and prognostic significance. Eur J Cancer 27:1461–1465
Song GX, Lin CT, Wu JY, Lam KW, Li CY, Yam LT (1985) Immunoelectron microscopic demonstration of prostatic acid phosphatase in human hyperplastic prostate. Prostate 7:63–71
Stein BS, Vangore S, Petersen RO (1984) Immunoperoxidase localization of prostatic antigens. Comparison of primary and metastatic sites. Urology 24:146–152
Trainer DL, Kline T, McCabe FL, Faucette LF, Feild J, Chaikin M, Anzano M, Rieman D, Hoffstein S, Li D-J, Gennaro D, Buscarino C, Lynch M, Poste G, Greig R (1988) Biological characterization and oncogene expression in human colorectal carcinoma cell lines. Int J Cancer 41:287–296
Warhol MJ, Longtine JA (1985) The ultrastructural localization of prostatic specific antigen and prostatic acid phosphatase in hyperplastic and neoplastic human prostates. J Urol 134:607–613
Wernert N, Seitz G, Achtstätter T (1987) Immunohistochemical investigation of different cytokeratins and vimentin in the prostate from the fetal period up to adulthood and in prostate carcinoma. Pathol Res Pract 182:617–626
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Riesenberg, R., Oberneder, R., Kriegmair, M. et al. Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells. Histochemistry 99, 61–66 (1993). https://doi.org/10.1007/BF00268022
Accepted:
Issue date:
DOI: https://doi.org/10.1007/BF00268022
