DETECTION OF TENASCIN-C ISOFORMS IN COLORECTAL MUCOSA, ULCERATIVE COLITIS, CARCINOMAS AND LIVER METASTASES Margret DUECK 1 , Stefan RIEDL 1 *, Ulf HINZ 1 , Andrea T ANDARA 1 , Peter MO ¨ LLER 2 , Christian HERFARTH 1 and Andreas FAISSNER 3,4 1 Department of Surgery, University Clinic of Heidelberg, Heidelberg, Germany 2 Department of Pathology, University Clinic of Heidelberg, Heidelberg, Germany 3 Institute of Neurobiology, Ruprecht-Karls-University, Heidelberg, Germany 4 Laboratoire de Neurobiologie du De ´veloppement et de la Re ´ge ´ne ´ration (LNDR, UPR 1352, CNRS), Centre de Neurochimie et Universite ´ Louis-Pasteur, Strasbourg, France The glycoprotein tenascin-C is up-regulated in inflamma- tory and neoplastic diseases. Most available data on tissue tenascin-C content do not distinguish its various isoforms. W e have quantified tissue tenascin-C signals in colorectal mucosa, ulcerative colitis, colorectal carcinomas and liver metastasesusing 5 monoclonal antibodies(MAbs) with differ- ent binding sites. T enascin-C of tissue extracts was analyzed by a standardized W estern blot technique and densitometry. Asa reference MAb, K8 displayed tenascin-C tissue concentra- tions of 4.1  2.3 g/mg total protein in normal mucosa, 13.8  4.7 g/mg in ulcerative colitis, 28.8  14.5 g/mg in colorectal carcinomas and 25.6  8.9 g/mg in liver metas- tases. The optical density values per microgram protein tissue extract of the 5 MAbs reflect the levels of the corre- sponding tenascin-C epitopes. Various signal intensities indi- cate a distinct diagnostic usefulness of the MAbs in detecting colorectal carcinomas. The binding characteristics of MAb J1/tn2 point to an under-representation of the T N fnD domain in metastasizing colorectal carcinomas, while MAb 19H12 showed an increased binding rate on the TNfnA1,2,4 region. Our comparative study of tenascin-C in inflammatory and neoplastic diseases of the colon mucosa substantiates the occurrence of large differences in the diagnostic value of tenascin-C MAbs. The detected alterations of tenascin-C in metastasizing colorectal carcinomasmight indicate a prognos- tic value of specific tenascin-C isoforms. Int. J. Cancer 82:477– 483, 1999.  1999 Wiley-Liss, Inc. The extracellular matrix component tenascin-C is a disulfide- linked hexameric glycoprotein. It is coded for by a single gene, which is composed of a serial arrangement of a cysteine-rich amino terminus, followed by 14.5 epidermal growth factor-like repeats and 8 to 16 fibronectin type III (FNIII) domains. The sequence is terminated by homology regions to the globular domain of  and -fibrinogen. Tenascin-C glycoproteins occur in various isoforms, which are created by the insertion of different combinations of alternatively spliced FNIII cassettes between the 5th and the 6th tenascin-C FNIII (TNfn) domains of the basic structure. Theoreti- cally, a large number of alternatively spliced isoforms can be generated. Of these, 9 have been described so far (Mighell et al., 1997). Substantial evidence has been provided that a large number of other isoform variants are realized and expressed in different tissues. It is likely that different tenascin-C isoforms reflect variations in biological function (Go ¨tz et al., 1996). During gut development in mouse embryos and human small intestine, a shift of molecular isoforms in correlation with developmental stages has been de- tected. In adult mouse and chicken tissue, the distribution of different tenascin-C mRNAs suggests tissue-specific expression of tenascin-C isoforms. Analogous observations have also been reported for human tissues. Tenascin-C is up-regulated under conditions of tissue remodel- ing in inflammatory processes and wound healing as well as during fetal and neoplastic growth. Within the colorectal mucosa, the luminal parts of the lamina propria and the basement membrane are tenascin-C-positive. In ulcerative colitis, tenascin-C is enriched in areas with increased tissue remodeling. In colorectal carcinomas, the tumor stroma and all parts of the basement membrane are homoge- neously tenascin-C-positive (Riedl et al., 1992; Sakai et al., 1993). High-m.w. variants of tenascin-C are enhanced in cancer tissues, e.g., in lung tumors, prostatic carcinomas, breast carcinomas, malignant ovarian tumors and CNS gliomas. In colorectal carcino- mas, a high-m.w. isoform of tenascin-C was detected in 7 of 15 tumors (Hauptmann et al., 1995). It was unknown whether certain combinations of alternatively spliced cassettes are preferentially up-regulated in different patho- logical disease entities of the large bowel. To investigate this question on the protein level, tissue extracts of normal colorectal mucosa, ulcerative colitis, colorectal carcinomas and their liver metastases were systematically examined for tenascin-C content with a panel of monoclonal antibodies (MAbs) which recognize separate binding sites. MATERIAL AND METHODS Tissue samples Tissue samples were taken from patients undergoing colon resection, resection of metachronous liver metastases or restorative proctocolectomy at the Department of Surgery, University of Heidelberg. Samples of 29 colorectal carcinomas were taken from patients with the following tumor staging (according to UICC): carcinomas without metastases (stage I/II), n = 10; carcinomas with lymph node metastases (stage III), n = 10; and carcinomas with synchronous distant metastases (stage IV), n = 9. Ten samples were obtained from surgically removed metachronous liver metas- tases of colorectal carcinomas. The 19 samples of colorectal mucosa with ulcerative colitis were obtained from patients with a clinical activity index according to Rachmilewitz (1989) which ranged from 1 to 17, with a median value of 5. Control samples of normal colorectal mucosa originated from 26 colon specimens removed in the surgical treatment of colorectal carcinoma and were obtained from macroscopically normal colon mucosa distant from the tumor. Informed consent was obtained from each patient included in the study, which had been approved by the Ethics Committee of the Heidelberg University Hospital. After surgical resection, fresh tissue was brought to our labora- tory within 2 hr. A pathologist experienced in gastrointestinal pathology (PM) obtained representative samples of the lesions without contamination of surrounding tissue and confirmed the histological diagnosis of all tissues prior to analysis of tenascin-C levels. All samples were snap-frozen in liquid nitrogen and stored at –70°C. MAbs MAbs K8 (diluted 1:100) (Scheffler et al. 1997), 20A1 (1:200), 19H12 (1:50) (C. Niederla ¨nder, personal communication), J1/tn1 Grant sponsor: German Research Council; Tumorzentrum Heidelberg/ Mannheim. *Correspondence to: Department of Surgery, University Clinic of Heidelberg, Im Neuenheimer Feld 110, D-69120 Heidelberg, Germany. Fax: +49 6221/564637. Received 4 September 1998; Revised 4 March 1999 Int. J. Cancer: 82, 477–483 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer