Establishment and characterization of a novel canine B-cell line derived from a spontaneously occurring diffuse large cell lymphoma

Leukemia Research 34 (2010) 932–938

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Establishment and characterization of a novel canine B-cell line derived from a spontaneously occurring diffuse large cell lymphoma Barbara C. Rütgen a,∗ , Sabine E. Hammer a , Wilhelm Gerner a , Maria Christian b , Abigail Guija de Arespacochaga b , Michael Willmann c , Miriam Kleiter c , Ilse Schwendenwein b , Armin Saalmüller a a

Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria Central Laboratory, Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria c Clinic for Companion Animal Medicine, Unit for Internal Medicine, Clinical Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria b

a r t i c l e

i n f o

Article history: Received 10 August 2009 Received in revised form 20 January 2010 Accepted 22 January 2010 Available online 11 February 2010 Keywords: Canine B-cell lymphoma Cell line Flow cytometry PARR

a b s t r a c t Cell lines derived from spontaneous tumors serve as a research tool for cancer cell biology and new anti-cancer drug development. Isolation and propagation of canine lymphoma cell lines is difficult, thus only a few are available. Now we have established a new B-cell lymphoma cell line CLBL-1 from a dog with confirmed stage IV diffuse large cell lymphoma. Immunophenotyping of these CLBL-1 cells showed positive staining for CD11a, CD79␣cy, CD45, CD45RA, MHC II and cells were negative for CD3, CD4, CD5, CD8, CD11d, CD14, CD21, CD34, CD56 and T-cell receptor-␥␦ (TCR-␥␦). PCR analysis for TCR-␥ and immunoglobulin heavy chain (IgH) gene rearrangements yielded a monoclonal result for the IgH gene. Furthermore, the clonality of IgH gene rearrangement was confirmed by sequencing of 16 positive bacterial clones. As canine lymphoma resembles non-Hodgkin’s lymphoma (NHL) in humans in many respects, this new cell line, will promote translational and comparative lymphoma research in humans and dogs. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Apart from skin and mammary gland tumors, lymphoma represents the most common spontaneously occurring tumor in dogs [1,2]. With 90% of all haematopoietic tumors [3] and 13–33 cases per year in 100,000 dogs [4], lymphoma has a much higher incidence in dogs than in humans. Dogs have shared a common environment with humans for thousands of years, thus dog cancers capture the ‘essence’ of human cancer in a unique manner which cannot be accomplished by any other animal model [5,6]. Spontaneously occurring canine lymphomas show strong similarities to high-grade non-Hodgkin’s lymphomas (NHLs) in humans with respect to histopathology, tumor genetics, disease progression and response to conventional therapies [7–12]. In contrast to humans, the remission time in canine lymphoma is much shorter. This compressed clinical course of disease reduces the time required to perform longitudinal studies. Furthermore, it is generally easier to test novel therapies at earlier time points in the course of disease than in human patients. All these facts underline the increasing

∗ Corresponding author. Tel.: +43 1 25077 2754; fax: +43 1 25077 2791. E-mail address: [email protected] (B.C. Rütgen). 0145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2010.01.021

importance of the dog as a spontaneous, clinically relevant, large animal model of non-Hodgkin’s lymphoma in humans. The heterogeneity of the disease regarding prognosis and therapy warrants differentiation of malignant tissue at the cellular level. Investigations into the aberrant molecular pathways present in canine lymphoma subtypes together with evaluation of novel therapies for this disease necessitate the availability of standardized and immunophenotypically characterized canine lymphoma cell lines with stable genotypes. Detailed investigations of canine lymphoma are hampered by the restricted availability of sufficiently characterized canine lymphoma cell lines. Primary cells reach senescence after a limited number of population doublings, therefore researchers frequently need to re-establish fresh cultures from explanted tissue. In order to use the same consistent material throughout a research project, primary cells with an extended replicative capacity in long-term cell culture or more preferably well characterized and widely distributed cell lines are required. In general, establishment of leukemia/lymphoma cell lines is known to be difficult [13]. Given the small amounts of sample material for the establishment of cell lines and the lack of specific growth factors for optimal cell culture conditions in many species, the establishment of cell lines in the dog is particularly challenging. There are few canine

B.C. Rütgen et al. / Leukemia Research 34 (2010) 932–938 Table 1 Monoclonal antibodies used for flow cytometry.

CD3 CD312 CD4 CD5 CD8 CD11a CD11d CD14 CD21 CD21-like (anti-B-cell) CD34 CD45 CD45RA CD56 CD79␣cy Thy-1 MHC II TCR␥␦

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2.2. Establishment of the canine B-cell lymphoma cell line CLBL-1

Clone

Isotype

Fluorescence labelling

CA17.2A12 CD3-12 YKIX302.9 YKIX322.3 YCATE 55.9 HI111 CA11.8H2 TÜK4 B-ly-4 CA2.106 1H6 YKIX716.13 CA4.1D3 MOC-1b HM57 YKIX337.217 YKIX334.2 CA20.8H1

mIgG1 rIgG1 rIgG2a rIgG2a rIgG1 mIgG1 mIgG1 mIgG2a mIgG1 mIgG1 mIgG1 rIgG2b mIgG1 mIgG1 mIgG1 rIgG2b rIgG2a mIgG2a

FITC FITC APC FITC PE APC anti-mouse IgG1-PEa anti-mouse IgG2a-PEa APC anti-mouse IgG1-PEa PE APC anti-mouse IgG1-PEa PE PE anti-rat IgG-PEa FITC anti-mouse IgG2a-APCa

Abbreviations: m = mouse; r = rat; FITC = Fluorescein isothiocyanate, APC = allophycocyanin; PE = phycoerythrin. a Fluorescence labelling was achieved by use of a secondary antibody. b Cross-reactivity pattern for canine lymphocytes [18].

lymphoma/leukemia cell lines described and even fewer well characterized and widely distributed. Most importantly, there is not a well described and characterized B-cell line established from the most common lymphoma histology in dogs and humans, the diffuse large B-cell lymphoma (DLBCL). To further accelerate the use of spontaneous cancers in dogs as a model system for spontaneously occurring cancer cell biology and new anti-cancer drug development, we established the B-cell lymphoma cell line CLBL-1 from a fine needle aspirate (FNA) of a dog with stage IV diffuse large cell lymphoma. The CLBL-1 cell line has been characterized by flow cytometry (FCM) and polymerase chain reaction for antigen-receptor rearrangement (PARR) representing now one of the rarely reported and well characterized canine lymphoma cell lines. Furthermore, three canine lymphoma/leukemia cell lines CL-1 [14], GL-1 [15] and OSW [16] were compared with the novel CLBL-1 cell line. Strikingly, these three cell lines have been established from liquid sample material acknowledging the CLBL-1 cell line as the first cell line of canine origin derived from a-FNA of a malignant peripheral lymph node. In our opinion, this diffuse large B-cell line will be a useful contribution to further research on canine lymphoma and will promote comparative lymphoma research in humans and dogs. 2. Materials and methods 2.1. Patient history, clinical diagnosis and outcome An 8-year-old male Bernese Mountain Dog was referred to the Clinic of Internal Medicine at the University of Veterinary Medicine Vienna in June 2008 for staging of a multicentric lymphoma. The dog had a 3-week history of lethargy and enlarged lymph nodes. At presentation the dog had generalized lymphadenopathy and abdominal enlargement, with palpable hepatosplenomegaly. Haematology revealed a low grade normocytic normochromic anaemia and marginal leukopenia with moderately low neutrophil- and lymphocyte-counts. Blood chemistry showed elevated alkaline phosphatase (20-fold) and gamma-glutamyl-transferase (6-fold) levels; urinalysis revealed marked proteinuria with a urine protein:creatinine ratio of 2.5. Abdominal ultrasound showed hepato- and spleno-megaly as well as marked enlargement of multiple intraabdominal lymph nodes. Thoracic radiographs showed enlargement of the sternal lymph nodes. One of the enlarged popliteal lymph nodes was sampled by core biopsy for histopathology and the other was sampled for FCM and tissue culture by FNA. Peripheral white blood cells (WBC) and the FNA of the popliteal lymph node were further analyzed by FCM. Monoclonal antibodies used for the analyses are presented in Table 1. Staining was performed as described below. Based on the World Health Organization Classification of lymphoma, a stage IV, diffuse large cell lymphoma was diagnosed. Stage V disease could not be excluded due to lack of adequate bone marrow samples. The dog was euthanized at the owner’s request.

0.5 × 107 cells from the FNA of the peripheral lymph node were cultured in RPMI 1640 medium (PAA, Pasching, Austria) supplemented with 20% heat inactivated fetal calf serum (FCS) (PAA) and penicillin 100 U/ml/streptomycin 0.1 mg/ml (PAA) at 37 ◦ C in a humidified atmosphere of 5% CO2 (T25-tissue culture flasks, Greiner Bio-One, Kremsmünster, Austria). Initially the cells, consisting of a mixed cell population derived from the lymph node without former selection of a certain population, showed mainly fibroblast-like, adherent appearance and were passaged every 4–5 days. After 2 months in continuous culture cells started to grow in free floating clusters, indicating the selective growth of round cells. Frequency of passage had to be increased to every 3 days. The CLBL-1 cell line has now been maintained in continuous culture for more than 11 months with over 105 passages. 2.3. Generation of growth curve CLBL-1 cells were seeded at a density of 1 × 105 cells in 4 ml of cell culture medium in 6 cm Petri-dishes (Greiner Bio-One). Cells were incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 and duplicate cultures were counted every 24 h for five consecutive days. Population doubling time (PDT) was calculated by the formula PDT = 1/[3.32(log NH − log NI )/(t2 − t1 )] (t1 = time in hours when cells were seeded; t2 = time in hours when cells were harvested; NI = cell count at time cells were seeded; NH = cell count at time cells were harvested) [17]. 2.4. Cloning of CLBL-1 CLBL-1 cells were seeded at a density of 1 and 3 cells per well in 96-well flat bottom culture plates (Greiner Bio-One). The cloning efficacy was determined by visual analyses [(% efficiency) = (colonies counted/cells inoculated) × 100]. Finally two clones were expanded and tested by FCM and PARR. 2.5. Former established cell lines used in this study The canine leukemia cell line GL-1 was published in 1996 [15]. It was derived from the peripheral blood of a 9-year-old neutered German Shepherd dog with acute B-cell leukemia at time of diagnosis. The CL-1 T-cell lymphoma cell line derived from the pleural fluid of a 7-year-old female Japanese Terrier with thymic lymphoma was published in 1997 [14]. The third cell line used in our studies – OSW – was published in 2007 [16]. It represents a T-cell lymphoma cell line from the malignant pleural effusion of a 5.5-year-old male Airedale Terrier with peripheral T-cell lymphoma after 3 weeks of chemotherapy. 2.6. Comparative cytology Morphology of the cultured cell lines was assessed by microscopic evaluation of cytospin preparations (stained with a modified Wright’s stain by an automated stainer (Haematek® , Bayer Diagnostics, Austria)). 2.7. Cell surface marker analyses WBC of the patient and the FNA were analyzed during routine diagnostic workup. These analyses were performed prior to cultivation of the FNA cells. After establishment of the CLBL-1 cell line, FCM experiments were repeated about every second month with passage P12, P23, P33, P50 and P67 to ensure the stability of the immunophenotype of the cells. For a comparison of CLBL-1 with the 3 former established cell lines CL-1, GL-1 and OSW their immunophenotype was determined in two separate experiments on exponentially growing cells at the same time. For all analyses cells were labelled with anti-canine or anti-human cross-reactive monoclonal antibodies (mAb) listed in Table 1. Most of these mAb were directly conjugated with fluorochromes (see Table 1 for details). For each analysis 5 × 105 cells were incubated with the listed mAb for 20 min on ice. After a washing step (PBS without Ca2+ and Mg2+ supplemented with 3% FCS), those samples containing mAb without directly conjugated fluorochromes were further labelled with anti-mouse secondary antibodies (as listed in Table 1) and incubated for an additional 20 min on ice. Finally, labelled cells were washed once again and analyzed on a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA) immediately after staining. For intracellular staining, the IntraStain-Kit (Dako, Glostrup, Denmark) was used according to manufacturers’ instructions. 2.8. Polymerase chain reaction for antigen receptor rearrangements (PARR) For the polymerase chain reaction for antigen receptor rearrangement (PARR) assay, total genomic DNA was extracted from FNA cells of the lymph node, 200 ␮l frozen blood as well as 5 × 106 cultured cells using a commercial kit following the manufacturers’ instructions (GenEluteTM Mammalian Genomic DNA Miniprep Kit; Sigma, Vienna, Austria) including negative extraction controls. The DNA was eluted with 200 ␮l elution buffer supplied with the kit. A 5 ␮l aliquot was analyzed on a 0.7% DNA grade agarose gel (Fisher Scientific, Schwerte, Germany) and visualized after staining with GelRedTM (Biotium, Hayward, CA, USA) together with a High

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Range DNA Ladder (GeneRulerTM High Range DNA Ladder, ready-to-use; Fermentas, Burlington, Ontario, Canada). The DNA samples were assayed by amplifying the C␮ DNA control [19], the immunoglobulin heavy chain (IgH) gene rearrangements with the primer sets CB1, CB2 and CB3 [19] and the T-cell receptor gamma (TCR␥) gene rearrangements with the primer sets DPA, DPB and DPC [16]. The PCR mixture was composed of 1× PCR buffer (KAPAHiFi High Fidelity Buffer with MgCl2 ; Peqlab, Erlangen, Germany), 0.2 mM of each nucleotide (PCR Grade; Peqlab), 10 pM of each primer (Eurofins MWG GmbH, Ebersberg, Germany), 1 Unit recombinant KAPAHiFiTM Proofreading DNA Polymerase (Peqlab) and 2 ␮l of a 1:10 dilution of eluted DNA as template brought up to 50 ␮l with molecular biology grade water (Sigma). Each PCR reaction was carried out in duplicate including positive and negative PCR controls in each PCR run. The PCR reactions were carried out using a T Gradient thermal cycler (Biometra, Göttingen, Germany) and the thermal cycling conditions were 95 ◦ C for 2 min, followed by 35 cycles at 98 ◦ C for 20 s, 60 ◦ C for 15 s and 68 ◦ C for 20 s. After PCR, the amplicons were first analyzed on a 4% low melting Phor agarose gel (Biozym Biotech Trading GmbH, Vienna, Austria) and visualized after staining with GelRedTM (Biotium) together with a 100-bp Ladder (O’GeneRulerTM 100 bp DNA Ladder, ready-to-use; Fermentas). To further investigate the clonality of IgH and TCR␥ gene rearrangements the obtained PCR products were cloned using the pJET1/blunt cloning vector (Fermentas) and 16 positive bacterial clones of each transformation were subjected to automated sequencing with pJET1 standard sequencing primers (Eurofins MWG GmbH, Ebersberg, Germany).

3. Results 3.1. Morphology and phenotype of primary lymphoma FNA cytology of the superficial cervical lymph node showed an infiltration of large lymphoid cells with multiple irregular nucleoli and finely stippled chromatin. The histological sample of the peripheral lymph node showed a diffuse architecture with colonization of the perinodal fat and few areas with small cell remnants. Artifact changes as well as necrotic areas in the sample hampered the visualization of mitotic figures. The nuclei of undamaged lymphoid cells were two small lymphocytes in diameter, with multiple prominent peripheral nucleoli. Architecture and cell morphology of representative cells were typical for a large cell lymphoma. FCM analyses of the FNA of the lymph node presented in Fig. 1b showed nearly 100% positive staining for CD3, CD79␣cy, CD21like (anti-B-cell), MHCII, CD45RA, CD11a and CD45 and weak to negative staining for CD11d and CD56. Some surface antigens showed a heterogeneous antigen expression with a small percentage of positive cells for CD4 (6.55%) and CD8 (8.5%, dim positive cells). Additional analyses presented in the Supplementary Table 1 revealed cells weakly positive for CD3-12, partially positive for CD5 (11.9%), CD21 and Thy1 (13.6%), but all cells were negative for CD14, CD34 and TCR-␥␦. White blood cells of the donor were analyzed in parallel to FNA-samples by FCM and showed the following percentages of positive cells (Fig. 1a, Supplementary Table 1): CD3 (58.5%), CD4 (29%), CD8 (34%), CD79 ␣cy (7.6%), CD21-like (anti-Bcell) (5.4%), MHC II (72%), CD11d (14.6%), CD45RA (72.4%), CD11a (all negative), CD45 (89%), CD56 (all negative). Additional markers presented in Supplementary Table 1 showed the following expression pattern: CD3-12 (69.1%), CD5 (76%), CD34 (all negative), CD56 (all negative) and Thy-1 (74%).

3.2. Establishment of the CLBL-1 cell line The CLBL-1 cell line was established after 2 months of continuous culture. The FNA cells grew without addition of any growth factors. CLBL-1 cells were arranged in small clusters of 10–20 cells and were small lymphoid cells showing minimal variation in nuclear cytoplasmic ratio. The nucleus was slightly eccentric with a dense chromatin structure and occasionally 3–5 marginating nucleoli were visible. The cytoplasmic rim was relatively small

Fig. 1. FCM analyses of primary WBC (a) and FNA (b). Cells derived from blood or FNA were labelled with various monoclonal antibodies against canine leukocyte antigens. In the top row dot-plots depicting forward/side scatter (SSC/FSC) signals are shown to illustrate gating of the respective lymphocyte populations. Histograms below show expression of various antigens of the gated lymphocyte population. Vertical lines in the histograms mark the boundaries between positive and negative cells for the respective markers as established by corresponding isotype control samples. Per sample at least 1 × 104 of gated cells were analyzed. WBC showed a heterogeneous antigen expression. FNA showed positive staining for CD3, CD79␣c␥, CD21-like (anti-B-cell), MHCII, CD45RA, CD11a and CD45.

and dark blue with a small pinkish vesicular halo near the nucleus (Fig. 2a). After 2 months of continuous passaging CLBL-1 cells showed a doubling time of 31 h during exponential growth under standard culture conditions. In general CLBL-1 cells grow in suspension, but after several months in culture adherent growth depending on the surface of the cell culture flasks and dishes can be observed. Over the time of cultivation CLBL-1 cells did not change their immunophenotype and antigen-receptor rearrangement which was confirmed by FCM and PARR, respectively. The cell line has now been maintained in continuous culture for more than 11 months through over 105 passages and grows in clusters (Fig. 2a) without supplementation of any growth factors.

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Fig. 2. Morphologic characteristics of the non-adherent cell lines CLBL-1 (a), GL-1 (b), OSW (c) and CL-1 (d) in vitro. In the upper row pictures of a cytospin preparation (Haematek® , original magnification 100×), in the lower row the pictures of invert light microscopy (original magnification 10×) are shown. The CLBL-1 and the CL-1 cells grow mainly in clusters whereas the GL-1 and the OSW show a growth pattern of single large, round cells.

3.3. Morphology and immunophenotype of the CLBL-1 cell line For phenotypical characterization five different passages of CLBL-1 cells, passage P12, P23, P33, P50 and P67, were analyzed. Cells derived from all passages analyzed so far showed the same phenotype. They were positive for CD79␣cy, MHC II, CD45RA, CD11a, CD45 and negative for CD3, CD4, CD5, CD8, CD11d, CD14, CD21, CD34, CD56 and TCR␥␦ (Fig. 3a, first column and Supplementary Table 2; data shown from one representative passage, P33). These results demonstrate a stable phenotype of the established cell line. The phenotypic data of P23, P33 (Fig. 3a) and P67 (Supplementary Fig. 1) show that the phenotype was already different and stable at P23 when compared to the FNA. Furthermore, after 6 month of continuous cell culture the CLBL-1 cell line was cloned with a plating efficiency of 24%. Two randomly chosen clones showed the same phenotype as the parental population in FCM and PARR (data not shown). 3.4. Clonal IgH gene rearrangement of the CLBL-1 cells PCR analysis of the CLBL-1 cells for TCR␥ and IgH gene rearrangements yielded a negative result for the TCR␥ gene and a single band for the IgH gene indicating a monoclonal result (Fig. 4c). In WBC and the FNA of the donor, positive results for the TCR␥ gene could be detected showing at least five autogenic clones. Furthermore, in DNA derived from FNA, IgH gene rearrangement could be found (Fig. 4a and b). In all samples, C␮ with about 130 bp served as a positive control [19]. The IgH products centered around 120 bp [19] and the TCR␥ products centered around 90 bp [16]. Sequencing analysis of 16 positive bacterial clones of each transformation confirmed the identical clonal rearrangement of the IgH gene for the CLBL-1 cells as well as the FNA sample. The respective DNA sequence was deposited in GenBank under accession no. FN295952. 3.5. Characterization and comparison of CLBL-1 with established cell lines In further analyses phenotype and receptor rearrangement of CLBL-1 was compared with data derived from previously published canine cell lines GL-1 [15], CL-1 [14], and OSW [16]. As described by the respective authors all these cell lines grew in suspension culture. GL-1 cells (Fig. 2b) grew in single cell suspension whereas the OSW and the CL-1 cells (Fig. 2c and d) grew as single cells and small clusters. As mentioned before, CLBL-1 grows in larger clusters (Fig. 2a). 3.5.1. Immunophenotyping For a further comparison of the phenotypes of the cell lines GL-1, OSW and CL-1 were stained with the same marker-panel used for

CLBL-1. Results are presented in Fig. 3. GL-1 cells (Fig. 3b) were positive for CD79␣cy, CD45RA, CD11a, CD45, partially positive for CD4 and CD11d (36.4% and 22.4%, respectively), and negative for CD3, CD8, CD21-like (anti-B-cell), MHC II, and CD56. OSW cells (Fig. 3c) stained positive for CD45 and CD11a and were partially positive for CD11d (23.2%), CD45RA (38.4%) and CD56 (30.3%). They were negative for CD3, CD4, CD8, CD79 ␣cy, CD21-like (anti-B-cell), and MHC II (Fig. 3c). The CL-1 cell line (Fig. 3d), also described as a Tcell line, was partially positive for CD79␣cy (80.5%) and CD21-like (anti-B-cell, 33.5%), and exhibited a uniform positive expression pattern for MHC II and CD45. This cell line did not express T-cell antigens as CD3, CD4, and CD8, and was furthermore negative for CD11d, CD45RA, CD11a, and CD56 (Fig. 3d). CL-1 cells were negative for the additional markers CD3-12, CD5, CD14, CD34, TCR␥␦ and Thy-1. (Supplementary Table 2.) 3.5.2. PARR analyses PCR analyses of the B-and T-cell receptor rearrangement of the four different cell lines revealed the following results. The OSW cells derived from a peripheral T-cell lymphoma displayed an oligoclonal TCR␥ gene rearrangement as described in the literature [16] (Fig. 4e). As expected, the cells of the T-lymphoblastoid cell line CL-1 [14] showed a monoclonal result for TCR␥ gene rearrangement (Fig. 4f). Interestingly, the cells of the B-cell leukemia cell line GL-1 [15] were positive for TCR␥ gene rearrangement instead of the expected IgH gene rearrangement (Fig. 4d) which is surprising considering their original characterization as B-cell line and the expression of distinct B-cell specific antigens, e.g. the high expression of CD79␣c␥. The CLBL-1 cell line showed a clear B-cell receptor rearrangement (Fig. 4c), whereas WBC and FNA of the donor of CLBL-1 (Fig. 4a and b) revealed a heterogeneous rearrangement pattern. The clonality of the gene rearrangements for the cell lines under investigation was confirmed by sequencing of 16 positive bacterial clones of each transformation. The respective DNA sequences were deposited in GenBank under the following accession nos. OSW: FN298232 and FN 298235; CL-1: FN298233; GL-1: FN298234. 4. Discussion In this study we describe the establishment of the novel canine lymphoma cell line CLBL-1 from a solid tumor of a popliteal lymph node of a dog with a confirmed stage IV diffuse large cell lymphoma. For investigations for comparative lymphoma biology and for new lymphoma treatments for translational research welldocumented and characterized canine lymphoma/leukemia cell lines for comparative studies and especially ones of B-cell character are required. To use the same consistent material throughout a whole research project is very important. Unfortunately, the establishment of leukemia/lymphoma cell lines is known to be difficult

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Fig. 3. Comparative FCM analyses of the cell lines CLBL-1 (a), GL-1 (b), OSW (c) and CL-1 (d) labelled with monoclonal antibodies against canine leukocyte antigens. In the top row dot-plots depicting forward/side scatter (SSC/FSC) signals are shown to illustrate gating of the respective cell populations. Histograms below show expression of various antigens of the gated cell populations. Vertical lines in the histograms mark the boundaries between positive and negative cells for the respective markers as established by corresponding isotype control samples. Per sample at least 5 × 104 of gated cells were analyzed. CLBL-1 cell line analyzed at different passages was positive for CD11a, CD79␣cy, CD45, CD45RA, MHC II, negative for CD3, CD4, CD5, CD8, CD11d, CD14, CD21, CD34, CD56 and TCR␥␦. GL-1 cells were positive for CD79␣cy, CD45RA, CD11a and CD45 and negative for MHCII, whereas OSW cells stained positive for CD11a and CD45 and negative for CD79␣cy, MHCII and CD45RA. The CL-1 cell line, an additional T-cell line, was positive for MHCII, CD45 and negative for CD79␣cy, CD45RA and CD11a.

Fig. 4. PCR for antigen receptor rearrangement. PARR of primary WBC (a) and FNA (b) and the cell lines CLBL-1 (c), GL-1 (d), OSW (e) and CL-1 (f). For each sample lane 1 shows C␮ which served as positive control for DNA. Lanes 2 and 3 show bands of TCR␥ and IgH PCR products, respectively. White blood cells and the FNA shared the same clones of the TCR␥ gene rearrangement. Only the FNA showed an additional monoclonal result for the IgH gene rearrangement. PCR analysis of the CLBL-1 cells yielded a monoclonal result (single band) for the IgH gene and a negative result for the TCR␥ gene rearrangements. The GL-1 and the CL-1 cells showed a monoclonal result for TCRy rearrangement and an oligoclonal TCR gene rearrangement was present in the OSW cell line. In all samples C␮ served as positive control [14].

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[13], and so detailed investigations of canine lymphoma are hampered by the restricted availability of such cell lines. Also in humans the success rates for leukemia/lymphoma cell line establishment are poor and depend on sampling site and treatment status. In humans, liquid sample material such as peripheral blood and pleural effusions show the highest success rate with 48% and 13%, respectively, whereas solid lymph node materials shows a success rate of only 3%. Actual treatment status also plays an important role. At relapse or in terminal stages the probability for successful cell line establishment is high (53%) whereas the feasibility at the time of diagnosis is only 35% and even much lower during therapy (2%) [13]. The canine CLBL-1 cell line was established at the time of diagnosis, prior to chemotherapy from solid lymphoid material taken from a dog with stage IV diffuse large cell lymphoma. B-cell origin of the cell line was confirmed by showing positive staining for CD11a, CD79␣cy, CD45, CD45RA and MHC II, and a monoclonal result for the IgH gene rearrangement. The sampling site and the characterization as DLBCL make these cells to the first well-characterized canine DLBCL line. The expression of MHCII in FCM suggests that this cell line exhibits an activated phenotype being typical for activated Bcells (ABC) DLBCL as compared to human [20,21]. Confirmation of this molecular subtype can be achieved by expression profiling analysis of candidate genes of the canonical NF␬B pathway, e.g. Bcl-2 and Bcl-XL. First attempts point towards an elevated expression level of the antiapoptotic Bcl-2 gene in CLBL-1, suggesting that this cell line may represent the ABC DLBCL phenotype. Furthermore, the case presented here, corroborates the importance of case history documentation and staging of patient material with various methods, not only for establishing cell lines but also for other lasting investigations in this research field. The fact that in this study, patient material, blood and FNA of a peripheral lymph node were available at the time of initial diagnosis made the data very comprehensive. FCM and PCR receptor rearrangement results of blood and FNA could be compared with several passages of the established cell line. The expression of CD3 and CD79␣c␥ as well as PARR analysis of the FNA led to the classification of diffuse large B-cell lymphoma with an aberrant phenotype. However, already the P23 of the established CLBL-1 cell line showed only a very weak expression of CD3 (Supplementary Fig. 1) and was negative for CD5 (Supplementary Table 2). Furthermore, CLBL-1 cells were negative for TCR␥ gene rearrangement identifying the established cell line as a diffuse large B-cell lymphoma. This phenotype was stable in all analyzed following passages (Supplementary Fig. 1). Therefore, if we suggest that at the start of the cultivation a diffuse large B-cell lymphoma with an aberrant phenotype existed, cultivation could have led either to the selection of a clone with B-cell characteristics (i.e. CD3− CD79␣c␥+ ) or to loss of the aberrant expression of T-cell surface antigens. Which one of these two scenarios took place during in vitro cultivation cannot be deduced from the data available. Another aim of the study was the comparison of the newly established cell line with other existing canine lymphoma lines: CL-1 [14], OSW [16] and Gl-1 [15]. More or less in accordance to the published results were our data with respect to the CL-1 T-cell lymphoma cell line [14]. Data from Momoi et al. could be confirmed, with the exception of the B-cell marker (CD21-like), which was 33.5% positive in our experiments. Additionally 11 monoclonal antibodies against a broad panel of antigens (CD4, CD14, CD21, CD45RA, CD11a, CD11d, CD34, TCR␥␦, CD3-12, CD79␣c␥, and CD56), which have not been tested before on CL-1 could contribute to a more detailed phenotypical description of this cell line. Originally, CL-l cells had a rearranged T-cell receptor ␤-chain gene and a germ-line form immunoglob-

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ulin gene, indicating that the CL-l cells represented a monoclonal population of canine ␣␤ T-cell lineage. This result was corroborated by the data of this study showing a monoclonal result for TCR␥ gene rearrangement characterising them unequivocally as T-cells. Another T-cell line was the recently published T-cell lymphoma cell line OSW. With the exception of CD45RA, which was negative in the publication and stained 38.4% of cells in our analyses, we were able to confirm the published phenotype as well as the oligoclonal TCR␥ gene rearrangement. The third published cell line compared with CLBL-1, the canine leukemia cell line GL-1 derived from a dog with acute B-cell leukemia had been tested in FCM for the expression of several antigens presented in Supplementary Table 2. In our studies these cells stained positive for CD45, and were negative for CD5 and Thy-1 confirming the results of the authors [15]. Interestingly the GL1 cells tested in our laboratory were negative for CD8 and CD14, whereas referring to Goto-Koshino Y. these cells should express these antigens. A further and more important kind of discrepancy to the original data was also shown by the result of the positive receptor rearrangement for TCR␥ characterising them as T-cells and not B-cells. These different results might be explained by (i) the usage of different clones of antibodies against canine CD8 and CD14 (Supplementary Table 2) or (ii) the possibility that GL-1 changed their phenotype over the long time since their establishment or (iii) that during the cultivation a sub-clone of GL-1 had been selected. Analyses of early passages of GL-1 might give an explanation for the differences in the respective results. In summary, the CLBL-1 B-cell lymphoma cell line, established from solid lymph node represents an interesting candidate for further investigations into the diagnosis and therapy of canine lymphoma. As canine lymphomas have many clinical and molecular similarities to NHL in humans, this cell line will additionally promote the translational and comparative lymphoma research in humans and dogs. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements Partial financiation for the study was provided by the “Hochschuljubiläumsstiftung” of the City of Vienna (Austria), project H-02307/2007 granted to S.E. Hammer. The authors are indebted to W.C. Kisseberth (Ohio State University, Columbus, USA) for providing the OSW and to H. Tsujimoto and Y. Goto-Koshino (University of Tokyo, Japan) for providing the CL-1 and the GL-1 cell line and to J.C. Duvigneau for the assistance in expression profiling analysis. The authors are also indebted to N.J. Mason and wish to thank her for carefully proofreading the manuscript. The helpful comments and valuable suggestions to improve the manuscript of two anonymous reviewers on the previous version of this paper are greatly acknowledged. Contributions. BCR, SEH and AS contributed in designing the research. MW and MK provided clinical care and management. MC, AGA and IS provided unique reagents, analysis, and interpretation. BCR, SEH and WG contributed to initial tumor cell line isolation and characterization. BCR, SEH and IS analyzed the data. BCR wrote the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.leukres.2010.01.021.

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