[CANCER RESEARCH 63, 4952–4959, August 15, 2003]

LABAZ1: A Metastatic Tumor Model for Renal Cell Carcinoma Expressing theCarbonic Anhydrase Type 9 Tumor Antigen

Amnon Zisman,1 Allan J. Pantuck, Matthew H. T. Bui, Jonathan W. Said, Randy R. Caliliw, Nagesh Rao,Peter Shintaku, Frank Berger, Sanjiv S. Gambhir, and Arie S. Belldegrun2

Division of Urologic Oncology, Department of Urology [A. Z., A. J. P., M. H. T. B., R. R. C., A. S. B.], Division of Anatomic Pathology [J. W. S., N. R.], Department of Pathology(Immunohistochemistry) [P. S.], Crump Institute for Molecular Imaging [F. B.], Molecular and Medical Pharmacology-Crump Institute [S. S. G.], David Geffen School ofMedicine at UCLA, Los Angeles, California

ABSTRACT

A metastatic renal cell carcinoma (RCC) tumor model xenograft thatexpresses the targetable, membrane-bound tumor-associated antigen car-bonic anhydrase type 9 (CA IX) is described. The xenograft, establishedfrom a high-grade type-2 chromophil RCC (cRCC), has been seriallytransplanted in immune compromised mice, in which it grows orthotopi-cally under the renal capsule, doubling its size every 9 weeks and sendingmetastases to the lung and liver at �20 weeks. Tumors were capable ofbeing imaged using a micro-PET (micro-positron emission tomograph)with an 18-fluorodeoxyglucose (18-FDG) tracer. Subsequent xenograftgenerations have conserved immunohistochemical and ultrastructuralproperties typical for malignant renal epithelium-derived neoplasia (vi-mentin�, CK-19�, CA IX� with hypoxia-inducible factor (HIF)-1� con-stitutive expression) and have demonstrated extensive proliferation, lackof apoptosis, severe genetic alterations, and molecular expression alter-ations; transforming growth factor �1 (TGF-�1), hepatocyte growth fac-tor (HGF), proto-oncogene (c-met), matrix metalloproteinase (MMP)-1,and vascular endothelial growth factor (VEGF) C and D were overex-pressed, whereas human epidermal growth factor receptor (HER)-2,MMP-2 and MMP-9, VEGF-R3, p53, and p27 were severely down-regu-lated, suggesting a proangiogenic environment, local invasiveness, andfacilitated lymphatic metastasis. Altogether, LABAZ1 provides a relevantand flexible model to study the biology of cRCC, the role of CA IX in RCCtumorigenesis, progression, and metastasis, and a platform for testing newtargeted therapeutic strategies.

INTRODUCTION

Chromophil (papillary) RCC3 is diagnosed in 15% of RCC, thesecond most frequent in occurrence after the clear cell type. cRCC hasbeen recognized as a distinct subtype of RCC based on histologicaland cytogenetic data (1). The existence of two different subtypes ofcRCC has been proposed (2, 3): type 1, having small cells with palecytoplasm and increased proportion of chromosome 7p and 17p gains(4); and type 2, having large eosinophilic cells, which occurs morecommonly in younger patients and which is associated with moreaggressive characteristics (3) and with worse prognosis (2, 4, 5).

CA IX is recognized as a type III biomarker (6), meaning that it is

still under investigation; it is induced constitutively in certain tumortypes but is absent in normal tissues with the exception of gastricepithelium. It is highly expressed in RCC, presumably because of aframeshift mutation in the von Hippel-Lindau (VHL) gene complex(7–12). CA IX is thought to have a role in the regulation of hypoxia-induced cell proliferation and may be involved in oncogenesis andtumor progression (7, 13, 14). We have recently shown that highexpression of CA IX is associated with improved prognosis in patientswith metastatic clear cell RCC (15) but is associated with poorprognosis in patients with nonhereditary type II cRCC.4 Such oppositerelationships between CA IX expression and prognosis have beenshown before: poorer in cervical carcinoma (16), colorectal carcinoma(17), and esophageal cancer (18), and improved survival in lungcancer and breast cancer (19).

Reports on the establishment of RCC cell lines (20, 21) or tumormodels (22–28) are available. However, the majority of these modelsare derived from cell lines and none are chromophil-type RCC or havemade the distinction between type 1 and type 2 subgroups. To developan orthotopic RCC xenograft tumor model in SCID mice that ex-presses the CA IX surface antigen, we have used tumor tissue from apatient with metastatic type 2 CA IX� cRCC. This model should beuseful for further investigation of the biology of type 2 cRCC and theimplication of CA IX expression, as well as for testing CA IX-targetedand other therapeutic modalities.

MATERIALS AND METHODS

Human Tumor. The primary human tumor originated from a 70-year-oldCaucasian female with a sporadic type 2 cRCC. The tumor was pathologicallystaged as T2N2M1 and graded 3 of 4 according to Fuhrman’s classification(Fig. 1). The patient received high-dose IL-2 immunotherapy 6 weeks afternephrectomy. At 3 years after treatment, the patient is alive with stable disease.

The specimen was opened under sterile conditions in the operating room.Fat, blood, and necrotic tissue were surgically removed. Small fragments of thefresh tumor tissue were cut and minced and reduced to a size of 2–4 mm andthen immersed in RPMI 1640 at 4°C and transferred on ice under sterileconditions to the SCID mice colony for immediate transplantation. The studywas approval by the Institutional Review Board (IRB) and the ExperimentalAnimal Committee of the University of California, Los-Angeles.

Animal Mode. Fifty male 6–8 week-old CB17 white SCID mice wereobtained from the breeding program at the Defined-Flora Pathogen-FreeMouse Colony at the University of California, Los Angeles, in five cohortsconsisting of 10 mice/cohort. The mice were kept in a pathogen-free isolatorunit. Food, water, and sawdust bedding were autoclaved, and the mice receivedchow and water ad libitum. The mice were anesthetized for survival surgerywith a mixture of ketamine (2 mg) and xylazine (0.6 mg; both from PhoenixScientific Inc., St. Joseph, MO) injected s.c. The site of incision was swabbedwith 70% ethanol solution. The right kidney was exposed through a right flankincision and was partially exteriorized. Using a 27-gauge needle, we injected100 �l of saline (0.9%) under the renal capsule to raise it from the underlying

Received 12/17/02; revised 5/23/03; accepted 6/15/03.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Present address: Department of Urology, Assaf Harofeh Medical Center, Tel AvivUniversity, Zerifin 70300, Israel.

2 To whom requests for reprints should be addressed, at Department of Urology, DavidGeffen School of Medicine at UCLA, Los Angeles, CA. 10833 Le Conte Avenue, CHS66-118, Los Angeles, CA. Phone: (310) 794-6584; Fax: (310) 206-5343; E-mail:abelldegrun@mednet.ucla.edu.

3 The abbreviations used are: RCC, Renal cell carcinoma; PET, positron emissiontomography; 18-FDG, 18-fluorodeoxyglucose; CK, cytokeratin; TGF-�1, transforminggrowth factor �1; HGF, hepatocyte growth factor; C-MET, Met proto-oncogene (HGFreceptor); MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor;HER-2, human EGFR-2; CA IX, carbonic anhydrase type 9; IL, interleukin; SCID, severecombined immune deficient; CD10, cluster of differentiation 10; TUNEL, terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling; EGFR, epidermal growthfactor receptor; bcl-2, B-cell lymphoma 2; PTEN, phosphatase and tensin homologue;STEAP, six-transmembrane epithelial antigen of the prostate; HIF, hypoxia-induciblefactor; cRCC, chromophil (papillary) renal cell carcinoma.

4 A. Zisman, M. Bui, A. Pantuck, J. Said, D. Seligson, F. Dorey, D. Chao, R. Figlin,and A. Belldegrun. Carbonic anhydrase IX expression is associated with lower survival oftype II non-hereditary chromophil (papillary) RCC—the UCLA experience, submitted forpublication.

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parenchyma. The injection resulted in the formation of a transparent subcap-sular vesicle. Then, a small slit was made on the vesicle using the tip of theneedle. The edge of the transparent elastic renal capsule was retracted on bothsides and 3–4 fresh tumor fragments were implanted into the subcapsularspace with 100 �l of Matrigel Matrix (Beckton-Dickenson Bioscience, Bed-ford, MA). The capsule was permitted to collapse and, therefore, fix the tumorfragments in close contact with the renal parenchyma. The kidney was thenallowed to fall back into the abdominal cavity. The body wall and the skinincision were closed separately with absorbable 5–0 vicryl sutures (EthiconINC, Somerville, NJ). Subsequent transfer of five xenograft generations wasperformed in the same manner. Immediate (1–3 days) postoperative mortalitywas 5 (10%) of 50. Animals were sacrificed at different time intervals, and theprimary tumor and organ of interest for metastatic spread were fixed andparaffin embedded.

Histology and Immunohistochemistry. Paraffin sections were cut at 3�m and were baked overnight at 60°C. Slides were deparaffinized in xyleneand rehydrated in graded ethyl alcohols series. Endogenous peroxidase activitywas eliminated by treating slides with 3% hydrogen peroxide in methanol for10 min. Heat-induced epitope retrieval was performed on the slides using 0.01M citrate buffer and steaming at pH 6.0 in a vegetable steamer for vimentin,keratin 19, and keratin 20, using 0.001 M EDTA (pH 8.0) and a pressure cooker(Biocare Medical, Walnut Creek, CA) for Ki67. After heating (25 min forvegetable steamer and 3 min for pressure cooker), cooling and washing in 0.01M PBS, slides were placed on a DAKO Autostainer (DAKO Corp, Carpenteria,CA) and then were sequentially incubated in primary antibody for 30 min,rabbit antimouse immunoglobulins (DAKO Corp) for 15 min, followed byEnvision� (rabbit, peroxidase; DAKO Corp) for 30 min. Diaminobenzidineand hydrogen peroxide were used as the substrates for the peroxidase enzyme.Primary antibodies included mouse monoclonal antibodies against vimentin(clone V9 at 1:1000 dilution), keratin 19 (clone RCK108 at 1:50 dilution),keratin 20 (clone K20.8 at 1:50 dilution), CD10, and Ki67 (clone MIB-1 at1:100 dilution). For negative controls, normal mouse serum was used in placeof the primary antibodies. All of the primary antibodies were obtained fromDAKO Corp. For detection of CA IX expression, sectioned tissue was incu-bated with primary antibody diluted 1:100. Slides were incubated with rabbitantimouse antibodies and then were detected with streptavidin horseradishperoxidase using the DAKO Envision� kit (DAKO). Normal murine kidneyserved as a negative control whereas a CA IX-transfected breast cancer cellline was used as a positive control. For HIF-1� staining, rabbit polyclonalantiserum specific for HIF-1� was obtained from Novus Biologicals Inc.,

Littleton, CO (Catalogue number NB 100-134). After antigen retrieval at115°C in TRIS buffer (pH 9.00), sections were incubated with primary anti-serum diluted at 1:100 to 1:250. Localization was performed with the DAKOEnvision� kit followed by horseradish peroxidase (DAKO Corporation). Thesections were counterstained with hematoxylin. For negative controls, normalrabbit serum was substituted for the primary antibody.

Sampled mouse xenograft tissues were fixed for 4 h in 10% bufferedformalin and were then embedded in paraffin blocks. Immunohistochemicalstaining was performed using the DAKO Animal Research Kit (ARK). Serialsections were subject to heat antigen retrieval, as described above, and wereincubated with the primary antibody diluted 1:100. The slides were incubatedsequentially with biotin-conjugated antimouse antibody with added normalmouse serum. Detection was performed with horseradish peroxidase using theARK kit. Cell proliferation was determined by Ki67 staining and was calcu-lated by manually counting total epithelial nuclei and Ki67-expressing epithe-lial nuclei in five microscopic fields in the primary human tumor, LABAZ1xenograft, and benign human or murine renal tissues to a total of 1000 nucleifor each. To assess apoptosis, we performed a TUNEL assay, as describedpreviously (29). For both the Ki67 and the TUNEL tests, results were testedusing ANOVA, with a P � 0.05 being considered significant.

Electron Microscopy. Tissue was obtained at the time of xenograft tumorresection and immediately fixed in gluteraldehyde for ultrastructural exami-nation. Tissue processing for ultrastructural examination was carried out usingmethods for the embedding of specimens for electron microscopy that havebeen described previously (30). Briefly, specimens were fixed in gluteralde-hyde, were postfixed for 1 h in osmium collidine, and were embedded in epon.

Cytogenetics. Chromosomal analysis was performed on fresh primary tu-mor and xenografts. On receipt, the tissue was aseptically minced with scalpelinto 2–3-mm pieces and was digested with collagenase II (5 mg/ml/100 gtissue; Worthington Biochem, Freehold, NJ), as described elsewhere (31, 32).After tissue dissociation, cells were washed and then were cultured in RPMI1640 supplemented with 20% fetal bovine serum and 1% penicillin/strepto-mycin at a final concentration of 1–2� 105 cells/5 ml of medium in a T-25flask. The cultures were harvested when growth was subconfluent and cellswere actively dividing. Cells were subjected to hypotonic treatment (potassiumchloride, 0.075 M) for 30 min at 37°C and were fixed in methanol and aceticacid (3:1). Chromosome analysis was performed on 20G-banding with trypsinand Giemsa (GTG)-banded metaphase cells from two or more cultures. Inter-pretation was made in accordance with International System for HumanCytogenetic Nomenclature (ISCN) guidelines (33). Numeric changes were

Fig. 1. Representative computerized tomography scanimage demonstrating the renal tumor (solid arrow) andretroperitoneal lymphadenopathy (empty arrow) in thepatient from whom LABAZ1 was derived.

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tabulated relative to specimen ploidy. Clonal abnormality was established onlywhen two or more cells had the same extra chromosome, structural abnormal-ity, or three or more cells with the same missing chromosome.

PET. Tumor-bearing mice were imaged using a PET scanner specificallydesigned for small animal imaging (micro-PET) with 18-FDG 96 h after tumortransplantation and once tumors were palpable. Mice were injected with200–300 mCi of radiolabeled tracer, placed in a spread supine position, andscanned with the micro-PET (34, 35). Scanning was performed 60–90 minafter tracer injection, to allow for clearance of background activity beforestarting image acquisition. Scanning was performed with the long axis of themouse parallel to the long axis of the scanner, with the mouse in a proneposition. Scanning conditions were identical to those described previously(36). Acquisition time was 56 min, 8 min/position, with seven bed positions.Images were reconstructed with filter-back projection and an iterative three-dimensional technique (37), with an isotropic image resolution of 1.8 mm anda volumetric resolution of �8 mm3.

Semiquantitative Reverse Transcriptase-PCR. Total RNA was extractedfrom the primary human tumor, from resected retroperitoneal lymph nodes,xenograft, contralateral normal murine kidney, LNCaP cell line, and CL1prostate cancer xenograft (38) as positive controls by acid guanidine isothio-cyanate-phenol-chloroform and were reverse transcribed into cDNA. Theamount of diluted cDNA that expressed equivalent signal intensity of �-actinwas used to perform PCR for the other markers. The oligonucleotide primerpair sequences and cycle settings used for �-actin; p53; p27; E-cadherin; IL-6and –8; VEGF-A, -B, -C, and -D; VEGF-R2 and -R3; EGFR; TGF-�1 and-�2; bcl-2; c-met; HGF; PTEN; STEAP; collagenase; MMP-2 and –9; andHER2 were as described previously (38, 39). The primer pair sequences forCA IX were 5�-CAA TAT GAG GGG TCT CTG ACTACA C and 3�-GGAATT CAG CTG GAC TGG CTC AGC A and the cycle settings consisted of94°C for 2 min followed by 30 cycles (94°C at 1 min, 65°C at 2 min).

RESULTS

Establishment of Tumor Model and Pattern of Growth

Forty-five (90%) of 50 mice survived the immediate postoperativeperiod. In the first xenograft generation, eight (80%) mice survived, ofwhich six grew palpable tumors (xenograft take rate, 75%). Startingwith LABAZ1 second generation, all of the mice surviving the post-

operative period grew tumors (xenograft transfer take rate, 100%). At96 h after subcapsular implantation of the xenograft particles, areduced 18-FDG uptake was noted over the right kidney but anincreased 18-FDG uptake was traced in the periphery of the rightkidney corresponding with the subcapsular location of the trans-planted LABAZ1 cells (Fig. 2).

The time to grow a 1-cm palpable tumor shortened progressivelywith each subsequent xenograft transfer (Table 1). One member ofeach xenograft generation cohort was sacrificed weekly after palpa-tion of 1 cm. Tumor, except for the final two mice in each cohort thatwere observed until the primary tumor grew to a size mandatingeuthanasia (Fig. 3). LABAZ1 tumors are locally invasive into neigh-boring organs (Fig. 4, A and B). On the basis of macroscopic organsurvey and histological examination of the lung, liver, bony skeleton,spleen, brain, and posterior peritoneal lymphatic tissue, the initialthree cohorts did not metastasize (Table 1). Starting with the fourthgeneration, metastases were noted at 19–20 weeks after xenograftimplantation. In generation 4 and 5, all of the mice sacrificed afterweek 19 and 20, respectively, had histological evidence of systemicmetastasis to the lung and/or liver as well as peritoneum, and all hadorthotopic tumors that have invaded into the liver as well as the smalland large bowel. For generations 4 and 5, time to grow a 2-cmpalpable tumor was 21 � 2 weeks, approximately corresponding withthe time to metastasis. Therefore, the doubling time of the tumor isestimated to be �9 weeks in the final LABAZ1 generation. Fourmice, kept on observation in generation 4 (n � 2) and 5 (n � 2), hadto be sacrificed for failure to thrive at 22 � 3 weeks, representing anapproximated survival of 23 weeks after xenograft implantation.

Histological and Immunohistochemical Characteristics

The original primary human tumor consisted of papillae lined byepithelial cells with abundant eosinophilic cytoplasm arranged in apseudostratified and irregularly stratified manner, compatible with theoriginal description of type 2 cRCC by Delahunt et al. (Ref. 3; Fig.5A). Typical for RCC, the primary tumor also reacted positively withantibodies for vimentin and CK-19 (Fig. 5, B and C, respectively) andnegatively for CK-20 (Fig. 5D). The primary human tumor waspositive for membranal CA IX (Fig. 5E), whereas murine tissues werenot (data not shown). This histological and immunohistochemicalprofile was reproduced in all LABAZ1 generations and shown forgeneration 5 only in a corresponding fashion in Fig. 5, F–J. Thepercentage of LABAZ1 xenograft cells staining positively for CD10was 5 � 1% (Fig. 5K) and for Ki67 36 � 5% in the primary humantumor and 58 � 3% in the LABAZ1 xenograft (P � 0.05; Fig. 5L).TUNEL staining did not demonstrate any significant apoptotic activ-ity in the LABAZ1 xenograft (Fig. 5M).

CA IX. The primary human tumor stained positive for CA IX (Fig.5E). The fifth generation LABAZ1 xenograft (Fig. 5J) and its meta-static lung (Fig. 5N) and liver deposits maintained a high level ofmembranal CA IX expression.

LABAZ1 xenograft particles were processed into single-cell sus-pension pooled and frozen at �180°C for 8 weeks. After beingdefrosted, LABAZ1 cells maintained membranal CA IX expression(Fig. 5O) and were tumorigenic both s.c. and orthotopically. The

Fig. 2. Micro-PET tomographic images using FDG as a PET reporter substrate of amouse xenografted with LABAZ1. Arrows, the xenografted right kidney.

Table 1 Generation of LABAZ1 RCC tumor model in four transfers of xenografts in SCID mice

Xenograftgeneration

Time to 1-cm palpabletumor (wk)

Time to metastasis(wk)

Mice survivingsurgery (tumor take)

Mice on observationsacrificed at (wk)

1 33 � 3 none 8 (6) 62 � 0.52 21 � 2 none 9 (9) 53 � 13 16 � 2 none 9 (9) 41 � 14 12 � 2 19 9 (9) 22 � 35 12 � 2 20 10 (10) 23 � 1

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immunohistochemical profile of these reconstituted xenografts wereidentical to fifth generation LABAZ1 xenografts.

HIF-1�. The primary human tumor stained positive for HIF-1�

(Fig. 5P). The fifth generation LABAZ1 xenograft and its metastaticlung metastasis (Fig. 5Q) maintained a high level of HIF-1� expres-sion, indicating that the CA-IX expression is attributable to the con-stituted expression of HIF-1 and is not caused by the effect of in vitrohypoxia.

Electron Microscopy

The LABAZ1 demonstrated clusters of malignant cells with ovalnuclei and nucleoli. The cytoplasm contained mitochondria, lipiddroplets, glycogen, and occasional lysosomes. The cell surface con-tained microvilli processes as well as intracellular lumens lined bymicrovilous processes indicating tubular differentiation, all of whichare typical for human malignant cells of epithelial origin (Fig. 6, A and

Fig. 3. LABAZ1 orthotopic xenograph (A) gross appearance after euthanasia. B, micro-PET all-body imaging of xenografted SCID mouse before euthanasia; compare with (C)normal mouse. Image visualized with FDG as PET reporter substrate. K, kidney; L, liver; X, xenograft.

Fig. 4. Local invasiveness and metastatic potential ofLABAZ1. A, a necropsy macroscopic view from theabdominal cavity to the chest demonstrating local exten-sion into the diaphragm, corresponding with B, vimentin-positive tumor deposits infiltrate the muscle layers of thediaphragm. C and D, H�E micrograph demonstratingpulmonary metastasis strongly expressing vimentin (D).E, H�E micrograph of macroscopically depicted livermetastasis seen on the liver surface away from the pri-mary tumor site. This lesion stains positive for vimentin(F). E1, inset, microscopically depicted liver metastasissituated inside the liver parenchyma.

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B). All of the LABAZ1 generations demonstrated the same ultrastruc-tural features (data not shown).

PCR results are summarized in Table 2. In comparison with thehuman primary tumor, LABAZ1 xenograft lost expression of HER-2,MMP-2 and MMP-9, VEGF-R3, p53, and p27 and had overexpressionof TGF-�1, HGF, c-met, MMP-1, and VEGF-C and -D, relatively, tothe primary human tumor and its lymph node metastases (except forTGF-�1).

Cytogenetic Analysis

The cytogenetic analysis of the primary human tumor showed a46XX female karyotype with an abnormal chromosome analysisconsistent with hypodiploid abnormal clones that have lost chromo-somes 1, 3, 4, 6, 9, 11, 13, 14, 15, 18, 21, and 22 with a 1:13translocation (34, X, �X, �1, �3, �4, �6, �9, �11, �13, der (13)t(1:13) (q21;q34), �14, �15, �16, �18, �21, �22 [cp18]/46, XX[2]). These features were consistent in the LABAZ1 xenografts except

that the xenograft demonstrated a jumping translocation of the shortarm of chromosome 1 from chromosome 13 in the primary tumor tochromosome 19 in the xenograft. The location of this translocationwas maintained in the ensuing LABAZ1 generations (Fig. 7).

DISCUSSION

We have established an orthotopic human RCC xenograft model inSCID mice that has been designated LABAZ1. The xenograft pro-duced tumor containing 46 chromosomes reproducibly, indicating it’shuman origin. Xenografts show ultrastructural features of malignantepithelial cells with Ki67 staining showing active proliferation andmarkedly low apoptotic activity. The expression of STEAP (40) alsosupports the malignant nature of LABAZ1 cells. Histologically andimmunohistochemically, all of the generations of LABAZ1 xenograftdemonstrated properties typical for human RCC such as CA IX,vimentin, and cytokeratins expression as well as local invasion and

Fig. 5. Histology of the primary human tumor. A, H�E micrograph, positive for vimentin (B) and CK-19 (C), negative for CK-20 (D), and positive for CA IX (E). F–J, thecorresponding stains for LABAZ1 xenograft. K, LABAZ1 CD10 staining; L, LABAZ1 Ki67 staining; M, TUNEL test for LABAZ1; N, LABAZ1 CA IX-positive staining of apulmonary metastasis; O, CA IX expression in LABAZ1 reconstituted single-cell suspension. Expression of HIF-1� in the primary human tumor (P) and in the LABAZ1 pulmonarymetastasis (Q).

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distant metastatic spread to the lung and liver. The ultrastructuralproperties of the LABAZ1 xenograft supports its origin from type 2cRCC. In a recent comparative study, Krishnan and Truong (41) havedemonstrated that “eosinophilic” cRCC is characterized by variablysized microvilli, small amounts of cytoplasmic lipid, and increasednumber of mitochondria in the cytoplasm, all compatible with ourTEM (transmission electron microscopy) findings in the LABAZ1xenograft, except that we have found glycogen deposits in theLABAZ1, whereas Krishnan and Truong suggested that the presenceof glycogen is typical for clear type RCC.

Molecular marker expression was also compatible with that previ-ously reported for cRCC (Table 2), including CD10 expression (42),down-regulation of HER-2 (43), and overexpression of MMP-1 withdown-regulation of MMP-2 and -9 (44). PTEN loss in clear cell RCC(45, 46) and allelic loss at 10q23.3 in chromophobe RCC (47) areconsidered a late-stage event and may contribute to the invasiveand/or metastatic tumor phenotype. Because LABAZ1 express PTEN,it is probable that PTEN does not have a role in the aggressiveness ofthis chromophil xenograft.

The tumorigenicity of RCC xenografts correlates with poor clinicaloutcome (23). The high xenograft take rate of LABAZ1 points to itsaggressiveness, which is further supported by overexpression of c-met. c-met has been recently shown to be associated with an aggres-sive phenotype in cRCC (48). LABAZ1 genetic instability, reflectedby major chromosomal alterations, are, by far, more extensive thenthose previously described for cRCC (49), indicating on aggressivebiological behavior. Other features that support aggressiveness areoverexpression of the proangiogenic and lymphangiogenic factorsIL-8 and VEGF-C and D, suggesting that tumor cells have thepropensity to metastasize via the lymphatics (50). Overexpression ofthe immune inhibitory molecule TGF-�1 and the expression of E-cadherin, IL-6, EGFR, VEGF-R2, VEGF-A and -B, TGF-�2, andbcl-2 are further reflected in the local invasiveness of LABAZ1, itsability to interfere with cellular immune responses, and its ability tosupport its growth with angiogenesis. LABAZ1 has low expression of

p27. Low p27 expression is reported to be a significant and independ-ent unfavorable prognostic factor in patients with RCC and to corre-late inversely with tumor aggressiveness (51). Conflicting evidencesas to the role of p53 expression and the biology of chromophil RCCexists in the literature (52–54). The LABAZ1 has low expression ofwild-type p53. Both p27 and p53 expression in the LABAZ1 xe-nograft support its biological aggressiveness and is in accord with thelow apoptotic activity of the xenograft.

Translocations of chromosome 1 have been reported previously forcRCC, e.g., a t(X;1)(p11;q21) translocation (55–59). Positional clon-ing has demonstrated that, as a result of this translocation, the tran-scription factor binding to IGHM enhancer 3 (TFE3) gene on theX-chromosome becomes fused to a novel gene, PRCC, on chromo-some 1. The small second exon of PRCC was found to be locatedadjacent to the t(X;1) breakpoint in the human gene on chromosome1 and be responsible for the production of a protein with an unknownfunction. It may be possible that the jumping translocation of chro-mosome 1 to chromosome 13 in the primary human tumor to chro-mosome 19 in the LABAZ1 xenograft may be of biological impor-tance and may exert a survival advantage for the xenograft. Furtherstudy is required to evaluate whether the association with chromo-some 19 at a given break point is a random phenomenon or is an eventof biological significance.

CA IX is the most significant tumor-associated molecular markerdescribed for kidney cancer to date. Its expression is associated withsignificant changes in tumor biology. An immunohistochemical studydemonstrated that CA IX is expressed in all types of RCCs, includingchromophil RCC but not in chromophobe histology nor in benignrenal lesions such as oncocytomas (12). We have recently shown thatCA IX expression is highly associated with survivorship for kidneycancer. Low CA IX expression predicts a worse outcome for patientswith locally advanced clear type RCC and is an independent predictorfor poor prognosis in patients with metastatic clear type RCC (15). In52 cRCC 65% were subclassified as type 2. The disease-specificsurvival of patients with type 2 chromophil RCC expressing CA IX,as an isolated group, was significantly inferior to those who were CA

Fig. 6. Transmission electron microscopy images of LABAZ1 xenograft generation 5.Images A2 and B2, the content in detail of the frames in micrographs A1 and B1,respectively. M, mitochondria; LD, lipid droplets; G, glycogen; MP, microvilli processes;IL, intracellular lumens.

Table 2 Summary of semiquantitative PCR of LABAZ1 xenograft sorted by expressionlevel relatively to the original primary human tumor and to the human lymph node

(LN) metastasis

Feature ExpressionRelative to human

primaryRelative to human

LN metastasis

HER-2 Negative 22 22MMP-2 Negative 22 22MMP-9 Negative 22 22VEGF-R3 Negative 2 2p53 Negative 22 2p27 Negative 2 22

TGF-�1 Positive 1 7HGF Positive 1 1c-met Positive 1 1IL-8 Positive 1 1MMP-1 Positive 1 1VEGF-C Positive 1 1VEGF-D Positive 1 1

TGF-�2 Positive 7 1bcl-2 Positive 7 7STEAP Positive 7 7VEGF-A Positive 7 7VEGF-B Positive 7 7PTEN Positive 7 1CD10 Positive 7 7

IL-6 Positive 2 7E-cadherin Positive 2 2CA IX Positive 2 2EGFR Positive 2 2VEGF-R2 Positive 2 22

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IX negative or type 1.4 Therefore, having a model that is CA IXpositive with constitutive HIF-1� expressing type 2 cRCC is impor-tant because it represents a platform to study an aggressive subtype ofchromophil RCC that, furthermore, expresses a targetable marker onits cell membrane.

The tumor-doubling time, time to metastasis, and estimated sur-vival of LABAZ1-bearing SCID mice are conserved within a narrowtime range. As such, it enables phenotypic characterization and thedevelopment and preclinical evaluation of possible therapeutic mo-dalities for metastatic RCC. Furthermore, the development of theLABAZ1 xenograft may be imaged in a micro-PET facility using18-FDG tracer as early as 96 h after implantation. The expression ofCA IX by the LABAZ1 can also be further exploited for noninvasive,RCC-specific molecular imaging using CA IX promoter activity (60)that drives the expression of reporter genes such as herpes simplexvirus thymidine kinase SR 39 (HSV-tk-SR39) for PET imaging (61) orluciferase for imaging with a charge-coupled device camera (62).Such constructs are currently being developed.

REFERENCES

1. Storkel, S., Eble, J. N., Adlakha, K., Amin, M., Blute, M. L., Bostwick, D. G., Darson,M., Delahunt, B., and Iczkowski, K. Classification of renal cell carcinoma: Work-group No. 1. Union Internationale Contre le Cancer (UICC) and the American JointCommittee on Cancer (AJCC). Cancer (Phila.), 80: 987–989, 1997.

2. Delahunt, B., Eble, J. N., McCredie, M. R., Bethwaite, P. B., Stewart, J. H., andBilous, A. M. Morphologic typing of papillary renal cell carcinoma: comparison ofgrowth kinetics and patient survival in 66 cases. Hum Pathol, 32: 590–595, 2001.

3. Delahunt, B., and Eble, J. N. Papillary renal cell carcinoma: a clinicopathologic andimmunohistochemical study of 105 tumors. Mod. Pathol., 10: 537–544, 1997.

4. Jiang, F., Richter, J., Schraml, P., Bubendorf, L., Gasser, T., Sauter, G., Mihatsch,M. J., and Moch, H. Chromosomal imbalances in papillary renal cell carcinoma:genetic differences between histological subtypes. Am. J. Pathol., 153: 1467–1473,1998.

5. Ono, Y., Ito, T., Tsujino, S., Aizawa, S., and Suzuki, M. A study of papillary renalcell carcinoma. Clinicopathological, immunohistochemical features and its typing.Nippon Hinyokika Gakkai Zasshi. Jpn. J. Urol., 88: 587–595, 1997.

6. Srigley, J. R., Hutter, R. V., Gelb, A. B., Henson, D. E., Kenney, G., King, B. F.,Raziuddin, S., and Pisansky, T. M. Current prognostic factors–renal cell carcinoma:Workgroup No. 4. Union Internationale Contre le Cancer (UICC) and the AmericanJoint Committee on Cancer (AJCC). Cancer (Phila.), 80: 994–996, 1997.

7. Ivanov, S., Liao, S. Y., Ivanova, A., Danilkovitch-Miagkova, A., Tarasova, N.,Weirich, G., Merrill, M. J., Proescholdt, M. A., Oldfield, E. H., Lee, J., Zavada, J.,Waheed, A., Sly, W., Lerman, M. I., and Stanbridge, E. J. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am. J.Pathol., 158: 905–919, 2001.

8. Zavada, J., Zavadova, Z., Pastorekova, S., Ciampor, F., Pastorek, J., and Zelnik, V.Expression of MaTu-MN protein in human tumor cultures and in clinical specimens.Int. J. Cancer, 54: 268–274, 1993.

9. Oosterwijk, E., Ruiter, D., and Joedemaeker, P. Monoclonal antibody G250 recog-nizes a determinant present in renal cell carcinoma and absent from normal kidney.Int. J. Cancer, 38: 489–494, 1986.

10. Murakami, Y., Kanda, K., Tsuji, M., Kanayama, H., and Kagawa, S. MN/CA9 geneexpression as a potential biomarker in renal cell carcinoma. Br. J. Urol. Int., 83:743–747, 1999.

11. Uemura, H., Nakagawa, Y., Yoshida, K., Saga, S., Yoshikawa, K., Hirao, Y., andOosterwijk, E. MN/CA IX/G250 as a potential target for immunotherapy of renal cellcarcinomas. Br. J. Cancer, 81: 741–746, 1999.

12. Liao, S. Y., Aurelio, O. N., Jan, K., Zavada, J., and Stanbridge, E. J. Identification ofthe MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of thekidney. Cancer Res., 57: 2827–2831, 1997.

13. Pastorek, J., Pastorekova, S., Callebaut, I., Mornon, J. P., Zelnik, V., Opavsky, R.,Zat’ovicova, M., Liao, S., Portetelle, D., Stanbridge, E. J., et al. Cloning andcharacterization of MN, a human tumor-associated protein with a domain homolo-gous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.Oncogene, 9: 2877–2888, 1994.

14. Wykoff, C. C., Pugh, C. W., Maxwell, P. H., Harris, A. L., and Ratcliffe, P. J.Identification of novel hypoxia dependent and independent target genes of the vonHippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling.Oncogene, 19: 6297–6305, 2000.

15. Bui, M., Seligson, D., Han, K., Pantuck, A., Dorey, F., Huang, Y., Horvath, S.,Leibovich, B., Chopra, S., Liao, S., Stanbridge, E., Lerman, M., Palotie, A., Figlin, R.,and Belldegrun, A. Carbonic anhydrase IX is an independent predictor of survival inadvanced renal clear cell carcinoma: implications for prognosis and therapy. Clin.Cancer Res., 9: 802–811, 2003.

16. Brewer, C. A., Liao, S. Y., Wilczynski, S. P., Pastorekova, S., Pastorek, J., Zavada,J., Kurosaki, T., Manetta, A., Berman, M. L., DiSaia, P. J., and Stanbridge, E. J. Astudy of biomarkers in cervical carcinoma and clinical correlation of the novelbiomarker MN. Gynecol. Oncol., 63: 337–344, 1996.

17. Saarnio, J., Parkkila, S., Parkkila, A. K., Haukipuro, K., Pastorekova, S., Pastorek, J.,Kairaluoma, M. I., and Karttunen, T. J. Immunohistochemical study of colorectaltumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX,with potential value as a marker of cell proliferation. Am. J. Pathol., 153: 279–285,1998.

18. Turner, J. R., Odze, R. D., Crum, C. P., and Resnick, M. B. MN antigen expressionin normal, preneoplastic, and neoplastic esophagus: a clinicopathological study of anew cancer-associated biomarker. Hum. Pathol., 28: 740–744, 1997.

19. Giatromanolaki, A., Koukourakis, M. I., Sivridis, E., Pastorek, J., Wykoff, C. C.,Gatter, K. C., and Harris, A. L. Expression of hypoxia-inducible carbonic anhydrase-9relates to angiogenic pathways and independently to poor outcome in non-small celllung cancer. Cancer Res., 61: 7992–7998, 2001.

20. Naito, S., Kotoh, S., Goto, K., Koga, H., Hasegawa, S., Noma, H., Yamasaki, T., andKumazawa, J. Establishment of two human renal cell carcinoma cell lines withdifferent chemosensitivity. Hum. Cell, 9: 101–108, 1996.

21. Shin, K. H., Ku, J. L., Kim, W. H., Lee, S. E., Lee, C., Kim, S. W., and Park, J. G.Establishment and characterization of seven human renal cell carcinoma cell lines. Br.J. Urol. Int., 85: 130–138, 2000.

22. Pulkkanen, K. J., Parkkinen, J. J., Kettunen, M. I., Kauppinen, R. A., Lappalainen,M., Ala-Opas, M. Y., and Yla-Herttuala, S. Characterization of a new animal modelfor human renal cell carcinoma. In Vivo, 14: 393–400, 2000.

23. Angevin, E., Glukhova, L., Pavon, C., Chassevent, A., Terrier-Lacombe, M. J.,Goguel, A. F., Bougaran, J., Ardouin, P., Court, B. H., Perrin, J. L., Vallancien, G.,Triebel, F., and Escudier, B. Human renal cell carcinoma xenografts in SCID mice:tumorigenicity correlates with a poor clinical prognosis. Lab Investig., 79: 879–888,1999.

24. Guha, A. K., Ghose, T., Singh, M., Aquino, J., Blair, A. H., Luner, S. J., andMammen, M. Tumor localization of monoclonal antibodies against human renalcarcinoma in a xenograft model. Cancer Lett., 61: 35–43, 1991.

25. Hillman, G. G., Droz, J. P., and Haas, G. P. Experimental animal models for the studyof therapeutic approaches in renal cell carcinoma. In Vivo, 8: 77–80, 1994.

26. Otto, U., Kloppel, G., and Baisch, H. Transplantation of human renal cell carcinomainto NMRI nu/nu mice. I. Reliability of an experimental tumor model. J. Urol., 131:130–133, 1984.

27. Otto, U., Huland, H., Baisch, H., and Kloppel, G. Transplantation of human renal cellcarcinoma into NMRI nu/nu mice. II. Evaluation of response to vinblastine sulfatemonotherapy. J. Urol., 131: 134–138, 1984.

28. Grossi, F. S., Zhao, X., Romijn, J. C., ten Kate, F. J., and Schroder, F. H. Metastaticpotential of human renal cell carcinoma: experimental model using subrenal capsuleimplantation in athymic nude mice. Urol. Res., 20: 303–306, 1992.

29. Cunha, G., and KD, V. Identification in histological sections of species origin of cellsfrom mouse, rat and human. Stain Technol., 59: 7–12, 1984.

30. Said, J., Chien, K., Tasaka, T., and Koeffler, P. Ultrastractural characterization ofhuman herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus) in Kaposi’s sarcoma

Fig. 7. Karyotype of the primary human tumorand LABAZ1 fifth generation. The jumping trans-location of chromosome 1 from chromosome 13 inthe primary tumor (arrow) to chromosome 19 (ar-row) is notable in the LABAZ1 xenograft. 1ry,primary.

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lesions: electron microscopy permits distinction from cytomegalovirus (CMV).J. Pathol., 182: 273–281, 1997.

31. Mohamed, A. N. and al-Katib, A. Establishment and characterization of a humanlymphoma cell line (WSU-NHL) with 14;18 translocation. Leuk Res, 12: 833–843,1988.

32. Mohamed, A. N., Mohammad, R. M., Koop, B. F., and al-Katib, A. Establishment andcharacterization of a new human Burkitt’s lymphoma cell line (WSU-BL). Cancer(Phila.), 64: 1041–1048, 1989.

33. Mitelman, F. ISCN (1995): An International System for Human Cytogenetic Nomen-clature. Basel: S. Karger, 1995.

34. Gambhir, S. S., Barrio, J. R., Phelps, M. E., Iyer, M., Namavari, M., Satyamurthy, N.,Wu, L., Green, L. A., Bauer, E., MacLaren, D. C., Nguyen, K., Berk, A. J., Cherry,S. R., and Herschman, H. R. Imaging adenoviral-directed reporter gene expression inliving animals with positron emission tomography. Proc. Natl. Acad. Sci. USA, 96:2333–2338, 1999.

35. Herschman, H. R., MacLaren, D. C., Iyer, M., Namavari, M., Bobinski, K., Green,L. A., Wu, L., Berk, A. J., Toyokuni, T., Barrio, J. R., Cherry, S. R., Phelps, M. E.,Sandgren, E. P., and Gambhir, S. S. Seeing is believing: non-invasive, quantitativeand repetitive imaging of reporter gene expression in living animals, using positronemission tomography. J. Neurosci. Res., 59: 699–705, 2000.

36. MacLaren, D. C., Gambhir, S. S., Satyamurthy, N., Barrio, J. R., Sharfstein, S.,Toyokuni, T., Wu, L., Berk, A. J., Cherry, S. R., Phelps, M. E., and Herschman, H. R.Repetitive, non-invasive imaging of the dopamine D2 receptor as a reporter gene inliving animals. Gene Ther., 6: 785–791, 1999.

37. Qi, J., Leahy, R., Cherry, S., Chatziioannou, A., and Farquhar, T. High-resolution 3DBayesian image reconstruction using the microPET small-animal scanner. Phys. Med.Biol., 43: 1001–1013, 1998.

38. Tso, C. L., McBride, W. H., Sun, J., Patel, B., Tsui, K. H., Paik, S. H., Gitlitz, B.,Caliliw, R., van Ophoven, A., Wu, L., deKernion, J., and Belldegrun, A. Androgendeprivation induces selective outgrowth of aggressive hormone-refractory prostatecancer clones expressing distinct cellular and molecular properties not present inparental androgen-dependent cancer cells. Cancer J., 6: 220–233, 2000.

39. Oda, Y., Sakamoto, A., Saito, T., Kinukawa, N., Iwamoto, Y., and Tsuneyoshi, M.Expression of hepatocyte growth factor (HGF)/scatter factor and its receptor c-METcorrelates with poor prognosis in synovial sarcoma. Hum. Pathol., 31: 185–192, 2000.

40. Hubert, R., Vivanco, I., Chen, E., Rastegar, S., Leong, K., Mitchell, S., Madraswala,R., Zhou, Y., Kuo, J., Raitano, A., Jakobovits, A., Saffran, D., and Afar, D. STEAP:a prostate-specific cell-surface anigen highly expressed in human prostate tumors.Proc. Natl. Acad. Sci. USA, 96: 14523–14528, 1999.

41. Krishnan, B., and Truong, L. D. Renal epithelial neoplasms: the diagnostic implica-tions of electron microscopic study in 55 cases. Hum. Pathol., 33: 68–79, 2002.

42. Avery, A. K., Beckstead, J., Renshaw, A. A., and Corless, C. L. Use of antibodies toRCC and CD10 in the differential diagnosis of renal neoplasms. Am. J. Surg. Pathol.,24: 203–210, 2000.

43. Latif, Z., Watters, A. D., Bartlett, J. M., Underwood, M. A., and Aitchison, M. Geneamplification and overexpression of HER2 in renal cell carcinoma. Br. J. Urol. Int.,89: 5–9, 2002.

44. Hagemann, T., Gunawan, B., Schulz, M., Fuzesi, L., and Binder, C. mRNA expres-sion of matrix metalloproteases and their inhibitors differs in subtypes of renal cellcarcinomas. Eur. J. Cancer, 37: 1839–1846, 2001.

45. Velickovic, M., Delahunt, B., McIver, B., and Grebe, S. K. Intragenic PTEN/MMAC1 loss of heterozygosity in conventional (clear-cell) renal cell carcinoma isassociated with poor patient prognosis. Mod. Pathol., 15: 479–485, 2002.

46. Brenner, W., Farber, G., Herget, T., Lehr, H. A., Hengstler, J. G., and Thuroff, J. W.Loss of tumor suppressor protein PTEN during renal carcinogenesis. Int. J. Cancer,99: 53–57, 2002.

47. Sukosd, F., Digon, B., Fischer, J., Pietsch, T., and Kovacs, G. Allelic loss at 10q23.3but lack of mutation of PTEN/MMAC1 in chromophobe renal cell carcinoma. CancerGenet. Cytogenet., 128: 161–163, 2001.

48. Sweeney, P., El-Naggar, A. K., Lin, S. H., and Pisters, L. L. Biological significanceof c-met over expression in papillary renal cell carcinoma. J. Urol., 168: 51–55, 2002.

49. Glukhova, L., Angevin, E., Lavialle, C., Cadot, B., Terrier-Lacombe, M. J., Perbal,B., Bernheim, A., and Goguel, A. F. Patterns of specific genomic alterations associ-ated with poor prognosis in high-grade renal cell carcinomas. Cancer Genet. Cyto-genet., 130: 105–110, 2001.

50. Stacker, S. A., Caesar, C., Baldwin, M. E., Thornton, G. E., Williams, R. A., Prevo,R., Jackson, D. G., Nishikawa, S., Kubo, H., and Achen, M. G. VEGF-D promotes themetastatic spread of tumor cells via the lymphatics. Nat. Med., 7: 186–191, 2001.

51. Migita, T., Oda, Y., Naito, S., and Tsuneyoshi, M. Low expression of p27(Kip1) isassociated with tumor size and poor prognosis in patients with renal cell carcinoma.Cancer (Phila.), 94: 973–979, 2002.

52. Ljungberg, B., Bozoky, B., Kovacs, G., Stattin, P., Farrelly, E., Nylander, K., andLandberg, G. p53 expression in correlation to clinical outcome in patients with renalcell carcinoma. Scand. J. Urol. Nephrol, 35: 15–20, 2001.

53. Vasavada, S. P., Novick, A. C., and Williams, B. R. P53, bcl-2, and Bax expressionin renal cell carcinoma. Urology, 51: 1057–1061, 1998.

54. Contractor, H., Zariwala, M., Bugert, P., Zeisler, J., and Kovacs, G. Mutation of thep53 tumour suppressor gene occurs preferentially in the chromophobe type of renalcell tumour. J. Pathol., 181: 136–139, 1997.

55. Weterman, M. A., Wilbrink, M., Janssen, I., Janssen, H. A., van den Berg, E., Fisher,S. E., Craig, I., and Geurts van Kessel, A. Molecular cloning of the papillary renal cellcarcinoma-associated translocation (X;1)(p11;q21) breakpoint. Cytogenet. Cell Gen-et., 75: 2–6, 1996.

56. Weterman, M. A., Wilbrink, M., Eleveld, M., Merkx, G., van Groningen, J. J., vanRooijen, M., and Geurts van Kessel, A. Genomic structure, chromosomal localization,and embryonic expression of the mouse homolog of PRCC, a gene associated withpapillary renal cell carcinoma. Cytogenet. Cell Genet., 92: 326–332, 2001.

57. Weterman, M. A., Wilbrink, M., and Geurts van Kessel, A. Fusion of the transcriptionfactor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11;q21)-positive papillary renalcell carcinomas. Proc. Natl. Acad. Sci. USA, 93: 15294–15298, 1996.

58. Weterman, M. A., Wilbrink, M., Dijkhuizen, T., van den Berg, E., and Geurts vanKessel, A. Fine mapping of the 1q21 breakpoint of the papillary renal cell carcinoma-associated (X;1) translocation. Hum. Genet., 98: 16–21, 1996.

59. Heimann, P., El Housni, H., Ogur, G., Weterman, M. A., Petty, E. M., and Vassart,G. Fusion of a novel gene, RCC17, to the TFE3 gene in t(X;17)(p11.2;q25.3)-bearingpapillary renal cell carcinomas. Cancer Res., 61: 4130–4135, 2001.

60. Kaluzova, M., Pastorekova, S., Svastova, E., J., P., Stanbridge, E., and Kaluz, S.Characterization of the MN/CA 9 promoter proximal region: a role for specificityprotein (SP) and activator protein 1 (AP1) factors. Biochem. J., 359: 669–677, 2001.

61. Pantuck, A. J., Berger, F., Zisman, A., Nguyen, D., Tso, C. L., Matherly, J., Gambhir,S. S., and Belldegrun, A. S. CL1-SR39: a noninvasive molecular imaging model ofprostate cancer suicide gene therapy using positron emission tomography. J. Urol.,168: 1193–1198, 2002.

62. Wu, J., Sundaresan, G., Iyer, M., and Gambhir, S. Noninvasive optical imaging offirefly luciferase reporter gene expression in skeletal muscles of living mice. Mol.Ther., 4: 297–306, 2001.

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2003;63:4952-4959. Cancer Res   Amnon Zisman, Allan J. Pantuck, Matthew H. T. Bui, et al.   Tumor AntigenCarcinoma Expressing the Carbonic Anhydrase Type 9 LABAZ1: A Metastatic Tumor Model for Renal Cell

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