Human Prostate Cancer in a Clinically Relevant Xenograft Mouse Model: Identiﬁcation of B(1,6)-Branched Oligosaccharides As a Marker of Tumor Progression

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 xenografted 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. 2012 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 2012 American Association for Cancer Research. patient (4). Apart from genetic models, human prostate

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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 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 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) / / 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 / / 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) tional tissue blocks. The embedded lungs were cut into 5- wasproventopredictmetastasisformationbothinclin- mm thick sections, every tenth section was retained and 10 ical and xenograft mouse studies of human breast and sections from the middle of the block were stained with colorectal cancer, therefore indicating the clinical rele- hematoxylin and eosin (H&E). Subsequently, pulmonary vance of the models (10, 11). metastases in these sections were counted using a light The aim of this study was to develop novel xenograft microscope, and the total number of lung metastases was mouse models for spontaneous metastatic human prostate quantified as described before (15). We furthermore deter- cancer and to furthermore provide evidence for its clinical mined circulating and disseminated tumor cells in a "proof- relevance by identifying a potential new prognostic glyco- of-principle experiment" with LNCaP-injected mice: after

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terminal anesthesia, murine blood and bone marrow were biochemical relapse (BCR). In all patients, PSA values were collected by cardiocentesis and by flushing the femora measured quarterly in the first year, followed by biannual diaphyses with NaCl 0.09%, respectively. DNA isolation measurements in the second and annual measurements kits were used to prepare genomic DNA from 200 mL mouse after the third year following surgery. Recurrence was blood and bone marrow suspension (QIAamp DNA Blood defined as a postoperative PSA of 0.2 ng/mL and rising Mini Kit; Qiagen) and concentrations of DNA were quan- thereafter; patients without evidence of recurrence were tified using a NanoDrop spectrophotometer (Peqlab). censored at last follow-up. All prostatectomy specimens Human tumor cells were quantified by real-time PCR using were analyzed according to a standard procedure. All pros- established primers specific for human Alu sequences as tates were completely paraffin embedded, including previously described (16). Numerical data were determined whole-mount sections as previously described (18). All against a standard curve established using murine blood H&E-stained histologic sections from all prostatectomy and bone marrow DNA containing log-fold dilutions of specimens were reviewed for the purpose of this study and DNA from 1 106 cell culture grown LNCaP cells (extracted one 0.6-mm thick tissue core was punched out from a using the QIAamp DNA Mini Kit; Qiagen). Negative con- representative cancer area and transferred onto a tissue trols included tissues obtained from noninjected mice. For microarray (TMA) format as described (19–22). For lectin each sample, analyzes were done in duplicates and as histochemistry, freshly cut TMA sections were transferred to independent experiments at least twice. xylol over night, deparaffinized and pretreated with 0.1% trypsin in lectin buffer (0.05 mol/L Tris-buffered saline, pH Immunohistochemistry and lectin histochemistry 7.6, with 1 mmol/L CaCl2 and MgCl2 added) for 15 minutes To confirm the human origin of metastatic deposits, at 37C. Afterwards, sections were incubated with biotiny- parallel sections to H&E-stained sections, in which at least lated PHA-L for 1 hour at room temperature. Slides were one metastasis was observed, were immunostained with then incubated with an avidin–alkaline phosphate complex human-specific monoclonal antibodies directed against (Vectastain, Vector, ABC Kit) for 30 minutes and lectin prostate-specific membrane antigen (Dako#M3620, Clone: binding was visualized as described before (17). PHA-L 3E6), a 65-kDa nonglycosylated protein component of binding was examined with regard to intensity (scaled mitochondria exclusively found in human cells (Millipor- from 1–3) and localization (cytoplasmic vs. apical/ e#MAB1273, Clone: 113-1) and HLA-1 (eBioscience#14- membranous). 9958, Clone: A4) by a standard procedure (14). Lectin histochemistry was done using biotinylated Phaseolus vul- Statistical analyses garis leukoagglutinin (PHA-L; Vector Labs) in a concentra- Statistical evaluations of the in vivo experiments were tion of 2 mg/mL as described before (17). done with GraphPad Prism software version 4. Glycosyla- tion array data were analyzed based on the DCt method Quantitative real-time PCR glycosylation array using an Excel-based data analysis template (SA Bioscience) RNA was extracted from cell culture grown LNCaP, PC-3, and were represented as x-fold up- or downregulation. The DU-145, and PPEC using a RNA isolation kit (RNeasy Mini PHA-L staining patterns on the TMA were statistically eval- Kit; Qiagen). LuCaP 23.1 xenograft tumors were not includ- uated using SPSS 19 software package (SPSS Inc.). Kaplan– ed, as they not only consist of human prostate cancer cells Meier curves were plotted for PSA recurrence analyses and but also of murine blood vessels, blood cells, connective compared using log-rank tests in a subset of patients with tissue, etc. RNA concentrations were quantified using a completed follow-up. Multivariate Cox proportional haz- NanoDrop spectrophotometer and 1 mg was processed to ard regression models were used for examination of com- cDNA with RT2 First Strand Kit (SA Biosciences). Quanti- peting risk factors. Student t, Fisher exact, Kruskal–Wallis, tative PCR analysis of 84 human glycosylation key genes and Mann Whitney U tests were done as appropriate to was assessed with the Human Glycosylation RT2 Profiler assess associations between variables. Statistical signifi- PCR Array (SA Biosciences) and RT2 SYBR Green qPCR cance was assigned at 2-tailed P values less than 0.05. master mix (SA Bioscience) in a LightCycler 480 (Roche). Five housekeeping genes and internal controls for genomic Results DNA contamination, RNA quality, and general PCR per- Spontaneous metastasis xenograft model formance were also included. The array was repeated twice Primary tumor growth was observed in at least 90% of with independently isolated RNA to generate statistically injected mice (Fig. 1A) and the termination criteria were relevant data. reached after a mean growth period of 72 (LNCaP), 47 (PC-3), 56 (LuCaP 23.1), and 77 (DU-145) days (Fig. 1B). Tissue microarray The growth period of PC-3 xenografts was significantly Prostate cancer specimens were available from 2,411 shorter than that of LNCaP and DU-145 xenografts patients, who underwent radical prostatectomy at the (P < 0.05). At necropsy, the average tumor weight was Department of Urology at the University Medical Center 1.48, 1.38, 1.29, and 0.87 g, respectively, without significant Hamburg-Eppendorf, Germany, between 1992 and 2005. differences between the cell lines (Fig. 1C). Pulmonary None of the patients received neoadjuvant endocrine ther- metastases were found in 100% of LNCaP- and PC-3– apy. Additional (salvage) therapy was initiated in case of a injected mice, in 50% of LuCaP 23.1 xenograft mice and

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A B C 100 100 * 2 80 1.5 60 50 1 40 20 0.5 Tumor take [%] Tumor weight Tumor weight [g] 0 [d] period Growth 0 0 LNCaP PC-3 LuCaP 23.1 DU-145 LNCaP PC-3 LuCaP 23.1 DU-145 LNCaP PC-3 LuCaP 23.1 DU-145 D E F 2,000 * 400 * 50 100 ** BM mL DTC/ 40 1,500 300 30 1,000 200 50 20 500 100 10 formation [%] CTC/ mL blood

Lung metastasis 0 0 0 0 Metastases per lung LNCaP PC-3 LuCaP 23.1 DU-145 LNCaP PC-3 LuCaP 23.1 DU-145 Control LNCaP Control LNCaP

H.E. H.E. H.E.

PaCNL 3-CP LuCaP 23.1

Anti-h-PSMA Anti-h-mitchondria Anti-HLA-1

20 µm

LNCaP LNCaP LNCaP

Figure 1. A–F, tumor growth and metastasis formation of 4 established human prostate cancer cell lines subcutaneously injected into pfp / /rag2 / mice. BM, bone marrow; bars represent mean SD, , P < 0.05, , P < 0.001. Photomicrographs (representative samples): top row, H&E-stained sections of mouse lungs containing prostate cancer metastases; bottom row, representative samples of immunohistochemical stainings of anti-human prostate–speciﬁc membrane antigen, anti-human mitochondria, and anti-HLA-1 antibodies in mouse lungs containing LNCaP metastases. The human origin of lung metastases was proven for each type of metastatic xenograft model (not shown for PC-3 and LuCaP 23.1). CTC, circulating tumor cells; DTC, disseminated tumor cells.

were not detectable in mice engrafted with DU-145 cells chemical staining of human prostate–specific membrane (Fig. 1D). The average total numbers of metastases were 548 antigen, mitochondria, and HLA-1 (Fig. 1). According to 503 (LNCaP), 1,387 1,171 (PC-3), and 74 1 (LuCaP Alu-PCR analyses, which were restricted to LNCaP-injected 23.1) per lung (Fig. 1E), so that the metastatic potential of mice, DNA of 266 circulating tumor cells (CTC) per mL the cell lines was classified as strong/moderate versus weak/ blood and of 35 disseminated tumor cells (DTC) per mL negative in the order PC-3/LNCaP versus LuCaP 23.1/DU- bone marrow was detectable (Fig. 1F). 145. According to statistical analyses, PC-3 xenograft mice developed significantly more lung metastases than LuCaP Mgat5b expression and PHA-L binding 23.1 and DU-145 xenograft mice (P < 0.05). The human On the basis of glycosylation quantitative real-time origin of lung metastases was confirmed by immunohisto- PCR (qRT-PCR) array data, the gene for b1,6-N-

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Average Ct Fold up- or downregulation Gene name PPEC LNCaP PC-3 DU-145 MGAT5B 33.85 8.56 26.63 * 18.39 FUT8 26.08 3.42 5.93 * 3.33 FUT11 26.29 1.7 4.42 * 3.14 GALNT13 34.95 31.6 31.16 -2.22 GALNT14 27.63 -84.74 -56.95 * 6.29 * EDEM1 24.06 -1.64 -4.86 * -3.09 ST8SIA6 31.55 -6.57 -18.11 2.63 GCNT4 27.97 -20.09 * -29.96 ** -2.27 * HEXA 28.17 -1.24 -107.51 * 1.43

50 µm 50 µm 20 µm LNCaP PC-3 PC-3 metastasis

50 µm 50 µm 50 µm DU-145 LuCaP 23.1 Normal human prostate

Figure 2. Glycosylation patterns and lectin binding of human prostate cancer cells. qRT-PCR analyses reveal an overexpression of Mgat5b (GnT-Vb, GnT-IX)in all cell culture grown prostate cancer cell lines. Expression levels were calculated based on the DCt method and represented as x-fold up- or downregulation compared with PPEC; , P < 0.05; , P < 0.001. Photomicrographs represent lectin histochemical staining with PHA-L, which speciﬁcally detects Mgat5b products, namely b1,6-branched oligosaccharides. Note the predominant presence of Mgat5b products in strongly/moderately metastatic xenograft tumors (PC-3 and LNCaP) and their lung metastases compared with weakly/nonmetastatic xenograft tumors (LuCaP 23.1 and DU-145) and normal human prostate.

acetylglucosaminyltransferase-5B (Mgat5b,alsoreported metastases. PHA-L binding was closely related to the as GnT-Vb or GnT-IX) was the single glycosyltransferase metastatic potential of the xenografts with the strongest gene consistently overexpressed in all 3 prostate cancer intensity in LNCaP and PC-3 primary tumors (and their cell lines compared with PPEC (Fig. 2). In detail, Mgat5b lung metastases) and the weakest intensity in LuCaP 23.1 expression was upregulated 8.5-fold in LNCaP cells, and DU-145 primary tumors (Fig. 2), suggesting PHA-L 18-fold in DU-145 cells, and 26-fold in PC-3 cells (P < as a helpful tool for metastasis prediction in our in vivo 0.05). As the products of the corresponding protein model. Moreover, we detected a differential translation Mgat5b, that is, N-linked b(1,6)-branched glycans, spe- of Mgat5b transcripts or differential regulation of Mgat5b ciﬁcally bind PHA-L (23, 24), PHA-L lectin histochem- activity in DU-145 cells and xenografts. PPEC and nor- istry was used to evaluate the Mgat5b activity in normal mal human prostate revealed almost absent Mgat5b human prostate, xenograft primary tumors, and lung expression levels and PHA-L binding, respectively.

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Table 1. Clinicopathologic features of the study populations

PHA-L–positive PHA-L–positive patients with patients (n ¼ 2,085) completed follow-up (n ¼ 1,446)

Patient age (y) Mean (median) 62.0 (62.0) 62.13 (62.0) Range 6.0–77.0 22.0–77.0 Follow-up (mo) Mean (median) 30.96 (26.17) 30.96 (26.17) Range 1.0–140.0 1.0–140.0 PSA (ng/mL) Mean (median) 9.4 (6.8) 10.1 (7.1) Range 0.0–102.8 0.0–102.8 Missing data 135 pat. (6.5%) 21 pat. (1.5%) pT stage No. of patients (percentage) pT2 1,339 (64.2%) 912 (63.2%) pT3a 373 (17.9%) 308 (21.3%) pT3b 218 (10.5%) 184 (12.7%) pT3c 25 (1.2%) 22 (1.5%) pT4 18 (0.8%) 17 (1.2%) pTx 112 (5.4%) 2 (0.1%) Prostatectomy Gleason score 6 906 (43.4%) 607 (42.0%) 3þ4 853 (40.9%) 658 (45.5%) 4þ3 189 (9.1%) 159 (11.0%) 8 27 (1.3%) 22 (1.5%) Missing data 110 (5.3%) 0 pN stage pN0 862 (41.3%) 784 (54.2%) pN1–3 56 (2.7%) 52 (3.6%) pNx 1,167 (56.0%) 610 (42.2%) Surgical margin status R0 1,567 (75.2%) 1,115 (77.1%) R1 407 (19.5%) 330 (22.9%) Rx 111 (5.3%) 1 (0.07%) Overall biochemical recurrences 318 (15.3%) 309 (21.4%)

Lectin histochemistry on a prostate cancer TMA tically not different with regard to their PSA recurrence-free As PHA-L binding was associated with the metastatic survival (data not shown). We furthermore examined, potential in our mouse model, we evaluated whether it was whether the localization of PHA-L was important for the also associated with diverging clinical courses of prostate clinical course of the PHA-L–positive patients as measured cancer patients using a TMA. Within our study population by postoperative PSA recurrences. Therefore, 639 patients of 2,411 patients, 2,085 (86.5%) were PHA-L positive and had to be excluded due to incomplete follow-up, resulting were subjected to further statistical analyses (clinicopatho- in a total of 1,446 spots (Table 1). The mean recurrence-free logic findings are summarized in Table 1). PHA-L binding survival was 95.8 (90.6–101.0; 95% CI) months. Most intensity was significantly correlated with the preoperative interestingly, a cytoplasmic binding of PHA-L adversely PSA values (available for 1,950 patients, Fig. 3A, Table 2, P ¼ affected PSA recurrence-free survival compared with api- 0.003, Kruskal–Wallis test). In addition, cytoplasmic stain- cal/membranous binding (Fig. 3B, Table 2, P ¼ 0.027, log- ing of PHA-L was significantly associated with a higher rank test). Unlike PHA-L localization, intensity had no preoperative PSA compared with apical/membranous stain- significant effect on PSA recurrence (Table 2). According ing (Fig. 3A, Table 2, P ¼ 0.017, Mann–Whitney U test). to multivariate analysis including all statistically relevant However, PHA-L intensity or localization did not correlate biochemical and clinical parameters (PSA, pT, pN, surgical with the Gleason score or tumor stage (data not shown). In margin status, Gleason, and age), PHA-L intensity and addition, the small subset of PHA-L–negative patients PHA-L localization were not independent predictors of PSA tended to lower PSA values, but these patients were statis- recurrence.

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A B ** 1.0 * 12 0.9 + 0.8 11 Apical/membranous 0.7 10 0.6 9 Mean PSA [ng/mL] Mean PSA Recurrence-free survival 0.5 8 Cytoplasmic P = 0.027 0.4 123Apical/ Cytoplas- membranous mic 0 24 48 72 96 120 144 PHA-L intensity PHA-L localization Months

Cytoplasmic Apical/membranous

Figure 3. PHA-L binding in prostate cancer patients. A, PHA-L binding intensity was significantly correlated with the preoperative PSA values (n ¼ 1,950, , P ¼ 0.023, , P ¼ 0.003). In addition, cytoplasmic staining of PHA-L was significantly associated with a higher preoperative PSA compared with þ apical/membranous staining ( , P ¼ 0.017). B, Kaplan–Meier curves showing PSA recurrence-free survival significantly declining from cytoplasmic to apical/ membranous binding of PHA-L (n ¼ 1,446). Photomicrographs showing cytoplasmic versus apical/membranous staining patterns of PHA-L in representative prostate cancer tissue microarray spots.

Discussion missed evidence for their clinical relevance (4). To over- As in practically all other solid neoplasms, formation of come these problems, we chose the pfp / /rag2 / mouse as distant metastases makes prostate cancer largely incurable the carrier of the human prostate cancer cells and showed a (25). To study metastasis formation, xenografted human considerable spontaneous pulmonary metastatic spread tumor cells in immunodeficient mice proved to be valuable after subcutaneous injection of prostate cancer cells. We tools to analyze mechanisms of metastasis in different additionally showed a multimodal dissemination of tumor tumor entities in general (13, 26). Yet, the prostate cancer cells into murine blood and bone marrow and confirmed field is critically lacking a metastasis xenograft model, in the human origin of lung metastases by immunohisto- which human tumor cells can be implanted in vivo and all chemical analyses. In contrast to previously used "leaky" the steps of the metastatic cascade are mimicked adequately SCID mice, pfp / /rag2 / mice are completely devoid of (27). Most of previous attempts used severe combined functional T- and B-lymphocytes as well as natural killer immunodeficient (SCID) mice and cumbersome proce- (NK) cells (30, 31). As NK cells are potent defenders against dures (6, 28, 29), bearing the risk of an artificial route of circulating tumor cells (14), the pfp / /rag2 / mouse metastasis delivery in some of these models (5). In contrast, strain is of particular interest in metastasis research. subcutaneous injections offer a valuable alternative with As aberrant glycosylation patterns of cancer cells have a lower risk of artificial dissemination. However, this been shown to be relevant for tumor progression in differ- approach was previously hindered by rare metastatic events ent tumor entities (9), we investigated the glycosylation in SCID-beige mice and difficult detection of the commonly profile of prostate cancer cells used in the present mouse micrometastatic lesions (6). Moreover, previous attempts model. We surprisingly identified an overexpression of (also including genetically "engineered" metastases) often Mgat5b in all prostate cancer cell lines compared with

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Table 2. PHA-L staining results on prostate cancer TMA and univariate analysis

PHA-L staining patterns and preoperative PSA-values (n ¼ 1,950)

No. of patients Mean PSA (ng/mL) P

Intensity 1 662 (33.9%) 8.8 2 914 (46.9%) 9.3 3 374 (19.2%) 10.6 0.003 Localization Membranous/apical 1,361 (69.8%) 9.1 Cytoplasmic 589 (30.2%) 10.0 0.017 PHA-L staining patterns and PSA recurrence-free survival (n ¼ 1,446)

No. of patients Mean recurrence-free survival (95% CI), mo P

Intensity 1 481 (33.3%) 103.0 (96.4–110.0) 2 661 (45.7%) 89.9 (81.0–99.0) 3 304 (21.0%) 89.9 (81.8–98.1) n.s. Localization Membranous/apical 977 (67.6%) 99.1 (91.6–106.6) Cytoplasmic 469 (32.4%) 85.3 (77.9–92.7) 0.027

normal prostate epithelium. The corresponding protein, transdifferentiation during malignant progression of pros- b(1,6)-N-acetylglucosaminyltransferase-Vb (also reported tate cancer. In addition to prostate cancer, increased as GnT-Vb or GnT-IX) was characterized as an isozyme of amounts of b(1,6)-branched oligosaccharides and corre- Mgat5 (GnT-V). Both enzymes synthesize the b(1,6)- sponding PHA-L binding are well-known predictors of poor GlcNAc linkage to a(1,6)-linked mannose on N-linked prognosis and decreased survival time in breast and colo- glycans, and the resulting carbohydrate structures of both rectal carcinomas (33, 36). Our results therefore widen the enzymes are specifically detected by the lectin PHA-L (32, significance of b(1,6)-branched oligosaccharides as markers 33). Mgat5b has so far exclusively been described in neural of tumor progression in human cancer. However, there are tissues and testes, whereas Mgat5 is expressed in most only few studies available about the underlying mechan- human and rodent tissues (23, 34). This work therefore isms, by which b(1,6)-branched oligosaccharides might describes increased expression levels of Mgat5b in human lead to tumor progression and metastasis formation in prostate cancer cells compared with primary human pros- prostate cancer. It is evident that increased b(1,6) branching tate epithelium for the first time (Fig. 2). On the basis of the significantly contributes to the malignant phenotype of presented mouse models (Fig. 1), the products of Mgat5b, melanoma cells by influencing their adhesion to extracel- namely b(1,6)-branched oligosaccharides, were predomi- lular matrix components and their motility (37). Increased nantly restricted to metastatic prostate cancer xenografts b(1,6)-branched N-glycans on b1 integrin stimulate the (PC-3, LNCaP; Fig. 2). In accordance with these findings, we migration of human fibrosarcoma cells in vitro (38), thus showed b(1,6)-branched oligosaccharides as markers of contributing to a more metastatic phenotype. In addition, tumor progression in prostate cancer patients (Fig. 3). different expression levels of Mgat5b potently regulate integ- However, PHA-L intensity and localization were indeed no rin and laminin-dependent adhesion and migration of independent predictors of recurrence-free survival (and human neuroblastoma cells in vitro (39). might be therefore not useful in clinical practice), but raise There are, however, different pathways by which Mgat5 the question of a mechanistic relevance of b(1,6)-branched and Mgat5b determine the malignant phenotype of cancer oligosaccharides in prostate cancer progression. cells by b1,6 branching of oligosaccharides. Mgat5b glyco- With regard to this question, it is of paramount interest sylates the neuron-specific receptor protein tyrosine phos- that the neuroendocrine transdifferentiation of prostate phatase b and by this causes higher levels of phosphorylated epithelium has already been noticed as a special feature of b-catenin, subsequently leading to decreased cell–cell adhe- human advanced prostate cancer (35). In this context, the sion and increased migration (40). In contrast to Mgat5b, predominance of the neuron-specific glycosyltransferase Mgat5 interacts with PTEN in an opposing manner and Mgat5b in metastatic xenografts and in prostate cancer of regulates growth factor signaling and cell polarity via PI3K/ patients with adverse prognostic markers in our study sup- Akt, leading to a more invasive malignant cell phenotype ports the pathophysiologic relevance of neuroendocrine (41). It has further been shown that cancer growth and

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Clinically Relevant Mouse Model of Human Prostate Cancer

metastasis formation of a spontaneous tumor model of (iiI) show a considerable amount of metastatic events that breast cancer (induced by the polyomavirus middle T (iii) reliably occur through the spontaneous metastatic oncogene) are reduced in mgat5 / mice (42). As expression cascade. Moreover, this is the first description of Mgat5b of Mgat5b in humans is restricted to the CNS and testes, expression and Mgat5b products in human prostate cancer. which are protected by a special blood–tissue barrier sys- tem, this enzyme represents a very interesting target for Disclosure of Potential Conflicts of Interest inhibitory treatment approaches (small molecules, miRNA, No potential conflicts of interest were disclosed. etc.), as prostate cancer cells are beyond this barrier and thus Acknowledgments directly and selectively targetable (43). The authors are thankful for the financial aid through the University In summary, this study describes a novel spontaneous Cancer Center Hamburg from the Deutsche Krebshilfe (Mildred Scheel Foundation) for the animal core facility. metastasis mouse model of human prostate cancer and by The costs of publication of this article were defrayed in part by the this identifies PHA-L as a marker of metastatic competence payment of page charges. This article must therefore be hereby marked in vivo and of tumor progression in prostate cancer advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. patients. We therefore suggest subcutaneous xenografts in / / pfp /rag2 mice as a suitable model of human prostate Received November 16, 2011; revised January 10, 2012; accepted January cancer, as we (i) can indicate its clinical relevance and 10, 2012; published OnlineFirst January 18, 2012.

References 1. YigitbasiO,OzturkU,GoktugHN,GucukA,BakirtasH.Prognostic 16. Nehmann N, Wicklein D, Schumacher U, Muller€ R. Comparison of two factors in metastatic prostate cancer. Urol Oncol 2011;29: techniques for the screening of human tumor cells in mouse blood: 162–5. quantitative real-time polymerase chain reaction (qRT-PCR) versus 2. Cheville JC, Tindall D, Boelter C, Jenkins R, Lohse CM, Pankratz VS, laser scanning cytometry (LSC). Acta Histochem 2010;112:489–96. et al. Metastatic prostate carcinoma to bone: clinical and pathologic 17. Thies A, Moll I, Berger J, Schumacher U. Lectin binding to cutaneous features associated with cancer-specific survival. Cancer 2002;95: malignant melanoma: HPA is associated with metastasis formation. Br 1028–36. J Cancer 2001;84:819–23. 3. Ahmad I, Sansom OJ, Leung HY. Advances in mouse models of 18. Schlomm T, Iwers L, Kirstein P, Jessen B, Kollermann€ J, Minner S, et al. prostate cancer. Exp Rev Mol Med 2008;10:e16. Clinical signifi cance of p53 alterations in surgically treated prostate 4. Valkenburg KC, Williams BO. Mouse models and prostate cancer. cancers. Mod Pathol 2008;21:1371–8. Prostate Cancer 2011;2011:895238. 19. El Gammal AT, Bruchmann€ M, Zustin J, Isbarn H, Hellwinkel OJ, 5. Bastide C, Bagnis C, Mannoni P, Hassoun J, Bladou F. A Nod Scid Kollermann€ J, et al. Chromosome 8p deletions and 8q gains are mouse model to study human prostate cancer. Prostate Cancer associated with tumor progression and poor prognosis in prostate Prostatic Dis 2002;5:311–5. cancer. Clin Cancer Res 2010;16:56–64. 6. Scatena CD, Hepner MA, Oei YA, Dusich JM, Yu SF, Purchio T, et al. 20. Fleischmann A, Schlomm T, Huland H, Kollermann€ J, Simon P, Mirla- Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal cher M, et al. Distinct subcellular expression patterns of neutral endo- model for the study of metastatic human prostate cancer. Prostate peptidase (CD10) in prostate cancer predict diverging clinical courses 2004;59:292–303. in surgically treated patients. Clin Cancer Res 2008;14:7838–42. 7. Namikawa R, Shtivelman E. SCID-hu mice for the study of human 21. Minner S, Enodien M, Sirma H, Luebke AM, Krohn A, Mayer PS, et al. cancer metastasis. Cancer Chemother Pharmacol 1999;43 Suppl: ERG status is unrelated to PSA recurrence in radically operated S37–41. prostate cancer in the absence of antihormonal therapy. Clin Cancer 8. Rosol TJ, Tannehill-Gregg SH, LeRoy BE, Mandl S, Contag CH. Animal Res 2011;17:5878–88. models of bone metastasis. Cancer 2003;97 3 Suppl:748–57. 22. Bubendorf L, Kononen J, Koivisto P, Schraml P, Moch H, Gasser TC, 9. Reis CA, Osorio H, Silva L, Gomes C, David L. Alterations in glyco- et al. Survey of gene amplifications during prostate cancer progression sylation as biomarkers for cancer detection. J Clin Pathol 2010; by high-throughout fluorescence in situ hybridization on tissue micro- 63:322–9. arrays. Cancer Res 1999;59:803–6. 10. Leathem AJ, Brooks SA. Predictive value of lectin binding on breast- 23. Kaneko M, Alvarez-Manilla G, Kamar M, Lee I, Lee JK, Troupe K, et al. A cancer recurrence and survival. Lancet 1987;1:1054–6. novel b(1,6)-N-acetylglucosaminyltransferase V (GnT-VB). FEBS Lett 11. Mitchell BS, Schumacher U. The use of the lectin Helix pomatia 2003;554:515–9. agglutinin (HPA) as a prognostic indicator and as a tool in cancer 24. Alvarez-Manilla G, Troupe K, Fleming M, Martinez-Uribe E, Pierce M. research. Histol Histopathol 1999;14:217–6. Comparison of the substrate specificities and catalytic properties of 12. Ellis WJ, Vessella RL, Buhler KR, Bladou F, True LD, Bigler SA, et al. the sister N-acetylglucosaminyltransferases, GnT-V and GnT-Vb (IX). Characterization of a novel androgen sensitive, prostate-specific anti- Glycobiology 2010;20:166–74. gen-producing prostatic carcinoma xenograft: LuCaP 23. Clin Cancer 25. Valastyan S, Weinberg RA. Tumor metastasis: molecular insights and Res 1996;2:1039–48. evolving paradigms. Cell 2011;147:275–92. 13. Thies A, Mauer S, Fodstad O, Schumacher U. Clinically proven mar- 26. Schumacher U, Adam E. Lectin histochemical HPA-binding pattern of kers of metastasis predict metastatic spread of human melanoma cells human breast and colon cancers is associated with metastases engrafted in scid mice. Br J Cancer 2007;96:609–16. formation in severe combined immunodeficient mice. Histochem J 14. Sodeur S, Ullrich S, Gustke H, Zangemeister-Wittke U, Schumacher 1997;29:677–84. U. Increased numbers of spontaneous SCLC metastasis in absence 27. Havens AM, Pedersen EA, Shiozawa Y, Ying C, Jung Y, Sun Y, et al. An of NK cells after subcutaneous inoculation of different SCLC cell in vivo mouse model for human prostate cancer metastasis. Neoplasia lines into pfp/rag2 double knock out mice. Cancer Lett 2009;282: 2008;10:371–80. 146–51. 28. An Z, Wang X, Geller J, Moossan AR, Hoffman RM. Surgical orthotopic 15. Jojovic M, Schumacher U. Quantitative assessment of spontaneous implantation allows high lung and lymph node metastatic expression of lung metastases of human HT29 colon cancer cells transplanted into human prostate carcinoma cell line PC-3 in nude mice. Prostate SCID mice. Cancer Lett 2000;152:151–6. 1998;34:169–74.

www.aacrjournals.org Clin Cancer Res; 18(5) March 1, 2012 OF9

Lange et al.

29. Stephenson RA, Dinney CP, Gohji K, Ordonez NG, Killion JJ, Fidler IJ. 36. Seelentag WK, Li WP, Schmitz SF, Metzger U, Aeberhard P, Heitz PU, Metastatic model for human prostate cancer using orthotopic implan- et al. Prognostic value of beta1,6-branched oligosaccharides in human tation in nude mice. J Natl Cancer Inst 1992;84:951–7. colorectal carcinoma. Cancer Res 1998;58:5559–64. 30. Shinkai Y, Rathbun G, Lam KP, Oltz EM, Stewart V, Mendelsohn M, 37. Przybylo M, Litynska A. Glycans in melanoma screening. Part 1. The et al. RAG-2-deficient mice lack mature lymphocytes owing to inability role of b1,6-branched N-linked oligosaccharides in melanoma. Bio- to initiate V(D)J rearrangement. Cell 1992;68:855–67. chem Soc Trans 2011;39:370–3. 31. Walsh CM, Matloubian M, Liu CC, Ueda R, Kurahara CG, Christensen 38. Guo HB, Lee I, Kamar M, Akiyama SK, Pierce M. Aberrant N-glyco- JL, et al. Immune function in mice lacking the perforin gene. Proc Natl sylation of b1 integrin causes reduced a5b1 integrin clustering and Acad Sci U S A 1994;91:10854–8. stimulates cell migration. Cancer Res 2002;62:6837–45. 32. Cummings RD, Kornfeld S. Characterization of the structural determi- 39. Abbott KL, Troupe K, Lee I, Pierce M. Integrin-dependent neuro- nants required for the high affinity interaction of asparagine-linked blastoma cell adhesion and migration on laminin is regulated by oligosaccharides with immobilized Phaseolus vulgaris leukoagglu- expression levels of two enzymes in the O-mannosyl-linked glyco- tinating and erythroagglutinating lecins. J Biol Chem 1982;257: sylation pathway, PomGnT1 and GnT-Vb. Exp Cell Res 2006;312: 11230–4. 2837–50. 33. Fernandes B, Sagman U, Auger M, Demetrio M, Dennis JW. b1-6 40. Abbott KL, Matthews RT, Pierce M. Receptor tyrosine phosphatase branched oligosaccharides as a marker of tumor progression in human beta (RPTPbeta) activity and signalling are attenuated by glycosylation breast and colon neoplasia. Cancer Res 1991;51:718–23. and subsequent cell surface galectin-1 binding. J Biol Chem 2008; 34. Inamori K, Endo T, Ide Y, Fujii S, Gu J, Honke K, et al. Molecular cloning 283:33026–35. and characterization of human GnT-IX, a novel b1,6-N-Acetylgluco- 41. Cheung P, Dennis JW. Mgat5 and Pten interact to regulate cell growth saminyltransferase that is specifically expressed in the brain. J Biol and polarity. Glycobiology 2007;17:767–73. Chem 2003;278:43102–9. 42. Granovsky M, Fata J, Pawling J, Muller WJ, Khokha R, Dennis JW. 35. Cantile M, Kisslinger A, Cindolo L, Schiavo G, D'Anto V, Franco R, et al. Suppression of tumor growth and metastasis in Mgat5-deficient mice. cAMP induced modifications of HOX D gene expression in prostate Nat Med 2000;6:306–12. cells allow the identification of a chromosomal area involved in vivo 43. Zhou X, Chen H, Wang Q, Zhang L, Zhao J. Knockdown of Mgat5 with neuroendocrine differentiation of human advanced prostate can- inhibits CD133 þhuman pulmonary adenocarcinoma cell growth in cers. J Cell Physiol 2005;205:202–10. vitro and in vivo. Clin Invest Med 2011;34:E155–62.

OF10 Clin Cancer Res; 18(5) March 1, 2012 Clinical Cancer Research

Human Prostate Cancer in a Clinically Relevant Xenograft Mouse Model: Identification of β(1,6)-Branched Oligosaccharides as a Marker of Tumor Progression

Tobias Lange, Sebastian Ullrich, Imke Müller, et al.

Clin Cancer Res Published OnlineFirst January 18, 2012.

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