Published OnlineFirst January 18, 2012; DOI: 10.1158/1078-0432.CCR-11-2900 Clinical Cancer Imaging, Diagnosis, Prognosis Research Human Prostate Cancer in a Clinically Relevant Xenograft Mouse Model: Identification of b(1,6)-Branched Oligosaccharides as a Marker of Tumor Progression Tobias Lange1, Sebastian Ullrich1, Imke Muller€ 1, Michael F. Nentwich1,2, Katrin Stubke€ 1, Susanne Feldhaus1, Christine Knies1, Olaf J.C. Hellwinkel3, Robert L. Vessella7, Claudia Abramjuk8, Mario Anders4, Jennifer Schroder-Schwarz€ 4, Thorsten Schlomm3,6, Hartwig Huland6, Guido Sauter5, and Udo Schumacher1 Abstract Purpose: To establish xenograft mouse models of metastatic and nonmetastatic human prostate cancer and to apply these models to the search for aberrant glycosylation patterns associated with tumor progression in vivo and in patients. Experimental Design: Prostate cancer cells (LNCaP, PC-3, LuCaP 23.1, and DU-145) were xeno- À À À À grafted subcutaneously into immunodeficient pfp / /rag2 / mice. Tumor growth and metastasis formation were quantified and as altered glycosylation patterns have been associated with metastasis formation in several other malignancies, prostate cancer cells were profiled by a quantitative real-time PCR (qRT-PCR) glycosylation array and compared with normal human prostate cells. The activity of upregulated glycosyltransferases was analyzed by their sugar residues end products using lectin histochemistry on primary tumors and metastases in the animal experiments and on 2,085 clinical samples. Results: PC-3 cells produced the largest number of spontaneous lung metastases, followed by LNCaP and LuCaP 23.1, whereas DU-145 was nonmetastatic. qRT-PCR revealed an upregulation of b1,6-N- acetylglucosaminyltransferase-5b (Mgat5b) in all prostate cancer cell lines. Mgat5b products [b(1,6)- branched oligosaccharides] were predominantly detectable in metastatic xenografts as shown by increased binding of Phaseolus vulgaris leukoagglutinin (PHA-L). The percentage of prostate cancer patients who were PHA-L positive was 86.5. PHA-L intensity correlated with serum prostate-specific antigen and a cytoplasmic staining negatively affected disease-free survival. Conclusion: We show a novel xenograft mouse model for human prostate cancer respecting the complete metastatic cascade. Specific glycosylation patterns reveal Mgat5b products as relevant markers of both metastatic competence in mice and disease-free survival in patients. This is the first description of Mgat5b in prostate cancer indicating a significant biologic importance of b(1,6)- branched oligosaccharides for prostate cancer progression. Clin Cancer Res; 18(5); 1–10. Ó2012 AACR. Introduction Authors' Affiliations: Departments of 1Anatomy and Experimental Morphology, 2General, Visceral and Thoracic Surgery, 3Urology (Sec- Prostate cancer is the most common malignancy in males 4 tion for Translational Prostate Cancer Research), Interdisciplinary and therefore considerably determining their overall mor- Endoscopy, and 5Pathology, University Cancer Center Hamburg; 6Martini-Clinic, Prostate Cancer Center, University Medical Center tality and life expectancy. Despite new therapeutic strate- Hamburg-Eppendorf, Hamburg, Germany; 7Department of Urology, gies, distant metastases still predict a poor prognosis with University of Washington Medical Center, Seattle, Washington; and limited time of survival (1, 2). Several attempts have been 8Department of Urology, University Hospital ChariteBerlin,Berlin, Germany made to model the metastatic cascade of prostate cancer metastases in mice, but most of these models were not T. Lange and S. Ullrich contributed equally to this work and therefore share successful. One of the main limitations so far remains the first authorship. induction of reliable spontaneous metastatic spread in a Corresponding Author: Tobias Lange, Department of Anatomy and model with proven clinical relevance. In short, a variety of Experimental Morphology, University Medical Center Hamburg-Eppen- dorf, Martinistrasse 52, 20246 Hamburg, Germany. Phone: 49-40-7410- genetically engineered metastases models in mice have 52591; Fax: 49-40-7410-5-5427; E-mail: [email protected] currently been developed (3), however, these investigate the mechanisms of progression of murine prostate cancer doi: 10.1158/1078-0432.CCR-11-2900 and do not necessarily reflect the situation in the human Ó2012 American Association for Cancer Research. patient (4). Apart from genetic models, human prostate www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst January 18, 2012; DOI: 10.1158/1078-0432.CCR-11-2900 Lange et al. sylation marker based on these models. By using subcuta- Translational Relevance neous xenograft models, we aimed to mimic the entire This is the first study investigating the prognostic effect metastatic cascade in a reproducible and easy-to-handle of b(1,6)-branched oligosaccharides recognized by their mouse xenograft model. specific lectin Phaseolus vulgaris leukoagglutinin (PHA-L) binding in human prostate cancer. The potential rele- Materials and Methods vance of these glycan structures was identified in newly established spontaneous metastasis xenograft models of Cell lines and culture conditions prostate cancer indicating PHA-L as a marker of meta- The LNCaP, PC-3, and DU-145 cell lines (all obtained static competence. To translate these findings into the from Deutsche Sammlung von Mikroorganismen und clinic, PHA-L binding was analyzed in tissue spots of Zellkulturen in 2007) and primary prostate epithelial cells 2,085 prostate cancer patients. As PHA-L was detectable (PPEC, obtained from American Type Culture Collection in in the vast majority of the patients, it can be suggested as 2010) were cultured according to the manufacturer’s pro- a potent marker of prostate cancer. Moreover, PHA-L tocol. For maintenance of LuCaP 23.1 cells (12), the pri- 3 binding significantly correlated with preoperative pros- mary tumors were resected when exceeding 2 cm or ulcer- ating the mouse skin, cut into small pieces and treated with tate-specific antigen (PSA) and adversely affected PSA recurrence-free survival, which supports our in vivo find- 0.05% Trypsin/EDTA (Gibco) at 37 C for 20 minutes. Cell ings and indicates the clinical relevance of our model. viability was determined by trypan blue counting, and only Although these results show a significant biologic impor- preparations with more than 95% viability were used for in tance of b(1,6)-branched oligosaccharides for prostate vivo injections (see below). cancer progression, the prognostic effect of PHA-L is not strong enough to add independent information to the Subcutaneous xenograft mouse model À À À À established clinicopathologic variables. Pfp / /rag2 / double knock out mice were generated by targeted mutation in C57BL/6 mice (B6.129S6- Pfptm1ClrkRag2tm1Fwa N12) and were obtained from Taco- nic. All mice were males aged 8 to 12 weeks and with a body cancer cell lines (such as LNCaP, PC-3, and DU-145) have weight between 20 and 25 g. Due to their immunodeficien- been used in subcutaneous, renal, and orthotopic xenograft cy, the animals were maintained under pathogen-free con- models. The latter ones are in so far limited, as orthotopic ditions in individually ventilated cages and fed with sterile 6 implantations of human tumor cells into the mouse pros- standard food and water ad libitum. A total of 1 Â 10 vital tate might bear the risk of an artificial route of metastasis LNCaP, PC-3, or DU-145 cells were resuspended in 200 mL delivery (5), although previous subcutaneous xenograft medium without supplements and were subcutaneously models rarely showed spontaneous metastatic spread (6). injected between the scapulae of narcotized (O2/CO2) À/À À/À Other xenograft approaches should rather be designated as pfp /rag2 mice (10 mice per cell line). The LuCaP "dissemination models" as they use intravenous injections 23.1 cell suspension of one primary tumor was transferred À/À À/À of tumor cells (7) or "metastasis microenvironment mod- to up to 3 pfp /rag2 mice using a Strauss cannule. All els" as they implant prostate cancer cells directly into the animal experiments were approved by the local animal mouse bone marrow as the site of metastasis (8), both of experiment approval committee and assigned to the project which do not reflect the entire metastatic cascade. Conse- number G08/75. quently, most of these previous attempts are the subject of debate with regard to their clinical and pathophysiologic Evaluation of tumor growth and metastasis formation relevance (4). When primary tumors exceeded 2 cm3 or ulcerated the The clinical relevance of xenograft mouse models was mouse skin, the mice were terminally anesthetized followed previously proven using the binding ability of specific by cervical dislocation. Primary tumors were excised, lectins toward defined carbohydrate structures on glyco- weighed, and processed for histologic examinations as proteins or proteoglycans. Aberrant glycosylation pat- described before (13, 14). Lungs were excised en bloc and terns, as detected by lectin histochemistry, have been fixed in formalin. After fixation, lungs were cut in 1-mm shown to be common events and hallmarks of cancer thick slices using razor blades, and the lung sections were progression for several tumor entities (9). In particular, embedded in agar and transferred to paraffin like conven- thebindingofthelectinHelix pomatia agglutinin (HPA)