New monoclonal antibodies against bilitranslocase 177 IzvIrNI zNaNstveNI člaNek New monoclonal antibodies against bilitranslocase 1 Department for the Production of Diagnostic Reagents and for Research, Blood Transfusion Center of Slovenia, Ljubljana 2 Department of Pathology, Izola General Hospital, Izola 3 Department of Systems Biology and Biotechnology, National Institute of Biology, Ljubljana * Authors contributed equally to this work, Ljubljana Korespondenca/ Correspondence: Uroš rajčević, uros.rajcevic@ztm.si Ključne besede: svetlocelični karcinom ledvičnih celic; bilitranslokaza; monoklonska protitelesa; UOk171 id 1548zdrav vestn | maj – junij 2017 | letnik 86 OnkologijaIzvirni znanstveni članek New monoclonal antibodies against bilitranslocase as a diagnostic tool in determining the progress of clear cell renal cell carcinoma Novi monoklonski protitelesi proti bilitranslokazi kot diagnostično orodje pri ugotavljanju napredovanja svetloceličnega karcinoma ledvičnih celic alexandra Bogožalec košir,*1,3 tjaša lukan,*1, 3 Mateja kukovec,2 vivijana snoj,2 sendi Montanič,1 vladka čurin Šerbec,1 Uroš rajčević1 Abstract Background: Monoclonal antibodies (mAbs) are an important tool in diagnostics and research, es- pecially when we are dealing with a protein marker of unknown primary structure as in the case of bilitranslocase (BTL). BTL is also expressed on kidney cells, where it acts as an organic anion transporter. We have shown earlier that there are differences in bilitranslocase expression in normal kidney cells versus early grade kidney cancer. Methods: We developed monoclonal antibodies against extra- and intra-cellular domains of bil- itranslocase protein model. To also gain a deeper insight in bilitranslocase expression in clinical samples, we assessed BTL expression in different grades of clear cell kidney cell carcinoma (ccRCC). Results: Both new monoclonal antibodies bind to a protein in UOK171 cells but not in the negative control. Binding of mAb is specific. mAb produced by cell line 2A9/2E9 (peptide 298–310; intracel- lular domain) is more suitable for immunohistochemical analyses as it gives stronger intensity of binding than mAb produced by cell line 11C9/2G9 (peptide 235–246; extracellular domain). Anti- body 2A9/2E9 stains bilitranslocase in proximal renal tubules of normal kidneys but not in the sur- rounding stroma. Staining decreases in grade I compared to normal kidney, gradually increases in grades II and III, and decreases again in grade IV of ccRCC tissue. Conclusions: Our results show that these antibodies can be used in different immunoassays. Fur- thermore, specificity and affinity of our mAbs allowed us to use them in the analysis of progressive grades of clear cell renal cell carcinoma in a limited number of patients. Thus, mAbs developed here can be used as a diagnostic tool that could help distinguish between early and late grades of clear cell renal cell carcinoma. Izvleček Uvod: Monoklonska protitelesa (mAbs) so pomembno orodje v diagnostiki in raziskovanju, še po- sebej kadar obravnavamo proteinski označevalec neznane primarne strukture, kot je v primeru bili- translokaze (BTL). BTL se izraža tudi na celicah ledvic, kjer deluje kot prenašalec organskih anionov. V prejšnjih študijah smo ugotovili, da se bilitranslokaza izraža različno v normalnih ledvičnih celi- cah v primerjavi z zgodnjimi fazami ledvičnega raka. 178 zdrav vestn | maj – junij 2017 | letnik 86 ONkOlOgIja Key words: clear cell renal cell carcinoma; bilitranslocase; monoclonal antibodies; UOk171 Citirajte kot/Cite as: zdrav vestn. 2017; 86:177–84. Prispelo: 17. 5. 2016 sprejeto: 17. 3. 2017 Metode: Razvili smo monoklonska protitelesa proti zunaj- in znotrajceličnim domenam proteinske- ga modela bilitranslokaze. Da bi pridobili globlji vpogled v izražanje bilitranslokaze tudi v kliničnih vzorcih, smo ugotavljali izražanje bilitranslokaze tudi pri različnih gradusih svetloceličnega karcino- ma ledvičnih celic (ccRCC). Izsledki: Obe novi monoklonski protitelesi se vežeta na protein v celicah UOK171, ne pa tudi v ne- gativnih kontrolah. mAb, ki ga proizvaja celična linija 2A9/2E9 (peptid 298–310; znotrajcelična do- mena), je bolj primeren za imunohistokemijske preiskave, ker daje večjo intenziteto vezave kot mAb, ki ga proizvaja linija 11C9/2G9 (peptid 235–246; zunajcelična domena). Protitelo 2A9/2E9 obarva bilitranslokazo v proksimalnih ledvičnih tubulih, ne pa v okoliški stromi. Obarvanje se zmanjšuje v gradusu I v primerjavi z normalno ledvico, postopno naraste v gradusih II in III ter ponovno pade v gradusu IV ccRCC. Sklepi: Naši rezultati kažejo na to, da se lahko ta protitelesa uporabijo pri različnih imunskih testih. Poleg tega sta nam specifičnost in afiniteta naših mAb omogočila njihovo uporabo pri analizi na- predovalih gradusov svetloceličnega karcinoma ledvičnih celic pri omejenem številu bolnikov. mAb, razvita v tej študiji, se lahko uporabijo kot diagnostično orodje za razlikovanje med zgodnjimi in poznimi gradusi svetloceličnega carcinoma ledvičnih celic. 1 Introduction In this work, we describe the use of anti-peptide monoclonal antibodies aga- inst bilitranslocase as a diagnostic tool. Bilitranslocase is a membrane protein localized in hepatocyte plasma mem- brane, basolateral domain of proximal tubules in the kidney, gastric epithelia and endothelial cells. Its main known biological purpose is to transfer organic anions from blood to hepatocytes (1,2,3). Although the primary structure of bi- litranslocase was predicted over a deca- de ago (4), a model of three-dimensional structure has been proposed only recen- tly (5), due to the low ability of membra- ne proteins to form a crystal structure and inability of isotopic reorientation in solution that might prevent structure solving using X-ray crystallography or NMR (5). Until recently, the most widely used antibody targeting bilitranslocase was an anti-peptide polyclonal antibody pro- duced by immunization of rabbit with a multi-antigen peptide, corresponding to segment 65–75 of the primary bilitran- slocase structure (4). In one of our recent studies monoclonal antibodies against segment 65–75 were described  (6) and in the present research monoclonal an- tibodies against segments 235–246 and 298–310 are characterized. According to the three-dimensional structure, both segments 65–75 and 235–246 belong to extracellular domains of bilitranslocase, crucial for its transport function, while segment 289–310 is an intracellular one and its function is yet unknown. In our previous research we were able to show that expression of bilitranslo- case is down-regulated in grade I clear cell renal cell carcinoma (ccRCC), using monoclonal antibodies against peptide 65–75 (6). Renal cell carcinoma (RCC) repre- sents 2 % of all cancers and is the seventh most commonly diagnosed cancer in men and the ninth most commonly di- agnosed cancer in women (7). It usually occurs in people between the ages of 50 and 70, where men are more exposed than women (8). Its incidence worldwi- de is about 300 000 new cases per year, a quarter of them present with advanced disease. RCC arises from the proximal renal tubular epithelium (9) and is a he- terogeneous disease made up of a num- ber of different histological types of can- New monoclonal antibodies against bilitranslocase New monoclonal antibodies against bilitranslocase 179 IzvIrNI zNaNstveNI člaNek cer  (10) that occur as an inherited and as a sporadic, non-inherited cancer (11). The most common histological diagnosis is a clear cell renal cell carcinoma (ccR- CC) which represents 85 % of kidney tu- mors  (12,13). The most studied form of inherited ccRCC is the one associated with von Hippel-Lindau (VHL) syndro- me  (11). Affected individuals are at risk for developing tumors in many organs, including the kidney. In VHL syndrome, one VHL allele, located on chromosome 3p25 and encoding for tumor suppres- sor protein is inherited with a mutation, when the other, wild-type VHL allele, is inactivated or silenced, and ccRCC ari- ses. Defects in the VHL gene also appear to be responsible for a high percentage of tumors in patients with sporadic ccR- CC (14,15). Recent high throughput sequencing projects have identified driver genes in ccRCC beyond VHL  (16,17,18). These studies identified frequently mutated tumor suppressors, including PBRM1, BAP1 and SETD2, all of which function as chromatin and/or histone modifiers and intriguingly map to the frequently lost 3p21 locus. New reports also suggest that additional pathogenic mechanisms are at play and suspect that ccRCC is a metabolic disease  (19), with the results potentially yielding novel therapeutic insights for the care of kidney cancer. Prognosis is closely related to the stage of disease  (9). The average life expectancy for patients whose disease is already advanced (locally invasive or metastatic renal-cell carcinoma) is about 13 months (9). Mortality rates of RCC are rising with its incidence worldwi- de of about 102 000 deaths per year (7). Therefore it is crucial to detect disease before it spreads, which is a major pro- blem, since in the early development the disease is characterized only by nonspe- cific symptoms (9,12,20). More specific symptoms such as flank pain, hematuria, and a palpable abdominal mass are signs of advanced disease (9). 2 Materials and methods 2.1 Cell culture Human clear cell renal carcinoma UOK 171 cell line was obtained at NIH- -NCI, Bethesda MD, USA. Cells were grown in Dulbecco modified Eagle’s me- dium (MP Biomedicals), supplemented with 10 % (v/v) fetal bovine serum, 1 % (v/v) glutamine, 0.34 % (v/v) penicillin (Sigma) and 0.34 % (v/v) streptomycin (Sigma) in a humidified incubator at + 37 °C and 5 % CO2. Cells were seeded to 6-well plates (1 × 105–4 × 105 cells per well) with round coverslips (2r = 13 mm), treated with Poly-L-Lysine (Sigma). Next day cells were ready for the immu- nocytochemistry assay. 2.2 Immunocytochemistry Cells were rinsed with PBS and fixed with ice-cold 70 % methanol (Sigma) in PBS for 20 min at +4°C. They were then permeabilized with 0.2 % Triton (Tech- nicon) in PBS for 10 min at room tem- perature (RT) and blocked with 4 % FCS in PBS for 15 min at RT. Samples were incubated with primary antibodies for 2h at RT. Cells were then incubated with appropriate fluorescent secondary anti- bodies Alexa Flour 555 Goat anti-Mouse IgG (Life Technologies), at concentrati- on 2 μg/ml in 4 % FCS and 0.1 % Triton in PBS for 1h at RT. Cell nuclei were stai- ned by Hoechst 33258, pentahydrate (bis- -benzimide) FluoroPure™ grade stain (Invitrogen), diluted 1:1000 in the same buffer as antibodies and incubated for 1 h at RT. Cells were washed three times with PBS between all incubation steps. After the final three washes with 4 % New monoclonal antibodies against bilitranslocase 180 zdrav vestn | maj – junij 2017 | letnik 86 ONkOlOgIja Fig. 1: Detection of bilitranslocase in UOk171 cells by monoclonal antibodies against peptides B (extracellular) and C (intracellular). specificity of binding of mabs 2a9/2e9 (peptide C) and 11C9/2g9 (peptide B) to bilitranslocase is confirmed by using negative control. Magnification bars: 10 μm. FCS and 0.1 % Triton in PBS, slides were mounted in Fluoromount (Sigma). Ne- gative controls were treated identically with omission of the primary antibody. The cells were visualized by fluorescent microscopy (Nikon, Eclipse Ti-E). 2.3 Immunohistochemistry Immunohistochemistry (IHC) was performed on paraffin embedded, archi- ved tissue sections of ccRCC and normal human kidney tissue sections. Immuno- histochemical analysis was performed on five samples (n=5) in triplicates for each grade  (21) of carcinoma, except grade 1, where only three samples (n=3) in triplicates were used. We also tested normal kidney samples. Two samples in five parallels were used. Briefly, tissue sections were deparaffinized in xylene, rehydrated in graded ethanol and trea- ted with sodium citrate buffer (20 mi- nutes at 95–100°C) for antigen retrieval. After washing in water and PBS, incuba- tion with undiluted primary antibodies in supernatant was performed at RT for 2h. Tissue slides were washed with PBS and treated with 2 % hydrogen peroxide (Sigma) in PBS for 8 min to block en- dogenous peroxidases, followed by incu- bation with secondary goat anti-mouse IgM antibodies (Sigma), conjugated with HRP (diluted 1:1000) for 2h at RT. After washing with PBS, reaction products were visualized by diaminobenzidine (DAB) as chromogen. Slides were rinsed with water. Mayer’s hematoxylin (Sigma) and Scott’s buffer (0.2 % (w/v) sodium bicarbonate, 2 % (w/v) magnesium sulfa- te) were used as a counterstain. Sections were dehydrated in graded ethanol fol- lowed by xylene before they were moun- ted with Eukitt mounting agent (Sigma) and inspected by pathologist for staining intensity, staining area, background and ccRCC grading. Negative controls were treated identically with omission of the New monoclonal antibodies against bilitranslocase New monoclonal antibodies against bilitranslocase 181 IzvIrNI zNaNstveNI člaNek Fig. 2: staining of bilitranslocase in four grades of human clear cell renal cell carcinoma (ccrCC) and normal human kidneys using mab 2a9/2e9. Magnification bar: 100 μm. primary antibody. Isotype controls were carried out by using monoclonal antibo- dies of the same antibody class against blood group antigen A. 2.4 IHC Statistics All diagnostic stainings were per- formed in triplicate. Negative controls were repeated five times. Pathologist inspected the IHC preparations for sta- ining intensity, staining area, backgro- und and ccRCC grading all by grading from 0–3. Mean and standard deviation of intensity values were calculated. Null hypothesis that there were no differences in mean values of staining intensities be- tween ccRCC grades, and ccRCC grades and normal kidney was tested by unpai- red (homoscedastic) two-tailed student’s t-test. The null hypothesis was rejected with p < 0.05. 3 Results 3.1 Binding of mAbs to BTL Antibodies were tested for their abili- ty to bind bilitranslocase in UOK171 cell line by fluorescent immunocytochemi- stry (Fig. 1). The results show that both monoclonal antibodies bind to a protein New monoclonal antibodies against bilitranslocase 182 zdrav vestn | maj – junij 2017 | letnik 86 ONkOlOgIja Fig. 3: staining intensities of the specimens of all four grades of ccrCC(gr.I- Iv) and normal human kidney tissue (gr. 0). Black lines above the histogram connect normal kidney with significantly differential grades in ccrCC. Blue lines above the histogram connect grade I with II-Iv ccrCC. asterisks above the connecting lines indicate the level of significance between the tested pairs. in UOK171 cells but not in the negative control. The binding of monoclonal an- tibodies is specific. 3.2 Applications of mAbs Based on the results of a pilot IHC test we concluded that mAb produced by cell line 2A9/2E9 (peptide 298–310; intracellular domain) is more suitable for immunohistochemical analyses as it gives stronger intensity of binding than mAb produced by cell line 11C9/2G9 (peptide 235–246; extracellular domain) and no background staining. Hence, this antibody was tested by immunohisto- chemical staining of bilitranslocase in all four grades of human clear cell renal cell carcinoma (ccRCC) and normal hu- man kidneys (Fig. 2). The results showed that our monoclonal antibody 2A9/2E9 stains bilitranslocase in proximal renal tubules of normal kidneys but not in the surrounding stroma. Figure 2 also shows that staining decreases in grade I, gradually increases in grades II and III, but then decreases again in grade IV of ccRCC tissue. To confirm the above-mentioned re- sults, we performed a statistical analysis of staining intensities in different gra- des of ccRCC and normal kidney (Fig. 3), n=5 for each grade, except grade 1 (n=3). Compared to normal kidney, bili- translocase staining intensity decreased in grade I ccRCC tissue as we reported before  (6). The difference however was not found to be significant (p = 0.086). Staining increases from grade I on and there is a significant difference between grades III and IV and normal kidney tis- sue. There is also a significant difference between grades II, III and IV and grade I of ccRCC. 4 Discussion For potential diagnostic purposes we selected two anti-peptide monoclo- nal antibodies against bilitranslocase (2A9/2E9 and 11C9/2G9, respectively). Antibodies are specific for extracellular domain crucial for BTL transport func- tion and C-terminal intracellular doma- in, respectively. They specifically bind both domains in the protein (Fig. 1, 2), enabling us to follow the expression of bilitranslocase in different tissues as well as in different cell lines according to the proposed model of BTL  (5). Specificity of antigen-antibody binding was charac- terized by different immunoassays (Fig. 1, 2). Moreover, to make sure our antibo- dies are specific for the targeted antigen, we introduced a set of isotype monoclo- nal antibodies (monoclonal antibodies of the same class, against another anti- gen) as a control, which confirmed our results as specific (Fig 1, 2). BTL expression in kidney cells was previously described in animal mo- dels (23) where it acts as a transporter of New monoclonal antibodies against bilitranslocase New monoclonal antibodies against bilitranslocase 183 IzvIrNI zNaNstveNI člaNek flavonoids from the diet. BTL expression was also demonstrated in normal human kidney and kidney cancer (6,23), where it was localized in distal tubules in normal kidney (6) and proximal and distal renal tubules while it was absent in renal corti- cal tumors (23). ccRCC originates in the epithelium of the proximal convoluted tubule, which transports glomerular fil- trate from the glomerulus to the descen- ding part of the nephron. In our study, we detected BTL expression in the epi- thelium of proximal tubules in normal kidneys but not in the surrounding stro- ma (Fig 2). We have shown earlier that BTL expression decreases in early grade ccRCC compared to normal kidneys (6) in a limited number of patients. To get a better insight in BTL expression in ccRCC and to demonstrate our novel monoclonal antibodies against bilitran- slocase as a tool in research and possibly in the diagnostics of this potential new biomarker, we performed this study on a larger number of kidney tumor samples from patients with progressive grades of ccRCC (stages I-IV) and normal kidney controls. We are able to confirm the dec- rease in BTL expression from normal kidney where it stained the proximal tubules to grade I ccRCC (Fig. 2, 3). The observed difference in BTL expression was statistically insignificant (Fig. 3). However, we were able to demonstrate that significant differences in bilitranslo- case exist as the cancer progresses from stage I towards stage IV (Fig. 3). There is also a significant difference between bili- translocase expression in normal kidney cells and grades III and IV of ccRCC. This way, based on BTL expression, we were able to distinguish between nor- mal kidneys and high grades (III-IV) and low- versus high-grade tumors (I vs. II-IV), while it was impossible to dis- tinguish between grades II-IV based on BTL expression. Why it appears that the BTL expression decreases in low-grade ccRCC compared to high-grade ccRCC remains elusive. The fact that ccRCC is suspected to be a metabolic disease (19) and that different genetic changes seem to be directed toward metabolic path- ways in our opinion this may be related to the disrupted transport and perhaps metabolism of antioxidants, where bili- translocase plays a role  (22). The exact role of bilitranslocase in the progression of ccRCC will have to be revealed by ad- ditional testing. 5 Conclusions In this work, specific monoclonal antibodies 2A9/2E9 and 11C9/2G9, of extracellular and intracellular C-termi- nal domains, respectively, of predicted primary structure of rat bilitranslocase were produced and characterized. De- scribed monoclonal antibodies could in future be used as a diagnostic tool that could help distinguish between early and late stages of clear cell renal cell carcino- ma. 6 Acknowledgements The authors gratefully acknowledge the help of Marjana Šprohar and Maja Černilec, BTCS in design and help in performance of immunoassays. Sabina Passamonti and Michela Terdoslavich are acknowledged for their help. This work was partially funded by European Regional Development Fund (Crossbor- der Cooperation Program Italy-Slove- nia 2007–2013, Trans2care project). The authors gratefully acknowledge the gift of UOK 171 cell line from dr. W. 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