A cell surface 230 kDa protein from murine melanoma involved with tumor malignancy

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Cancer Letters 262 (2008) 276–285

www.elsevier.com/locate/canlet

A cell surface 230 kDa protein from murine melanomainvolved with tumor malignancy

Priscila Fraga Penteado Mendes a, Patrıcia Xander a,Ronni Romulo Novaes e Brito a, Renato Arruda Mortara a,

Miriam Galvonas Jasiulionis a,b, Jose Daniel Lopes a,*

a Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sao Paulo,

Rua Botucatu, 862, 4� andar, Sao Paulo 04023-901, Brazilb Departamento de Farmacologia, Universidade Federal de Sao Paulo, UNIFESP, Sao Paulo, Brazil

Received 5 October 2007; received in revised form 4 December 2007; accepted 5 December 2007

Abstract

Because melanoma incidence has increased at a dramatic rate, it is relevant to identify novel melanoma antigens fordiagnosis and develop monoclonal antibodies recognizing such molecules. Some monoclonal antibodies (mAbs), raisedagainst murine melanoma, identify molecules correlated with carcinogenesis. Herein, we describe a murine melanoma-associated 230 kDa molecule, expressed only in tumorigenic cell lines. Moreover, its expression is higher in more metastaticthan less metastatic cells. G12F2 mAb, produced against this antigen, inhibited in vitro proliferation of both murine andhuman melanoma cells and enhanced in vitro complement activity. It also affected in vivo tumor growth and lung metas-tases formation. This 230 kDa molecule represents an important target for experimental melanoma studies and maybecome a potential diagnostic marker for malignancy as well as a useful tool for immunotherapeutic approaches.� 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Murine melanoma; Monoclonal antibody; Tumor marker; Malignancy; Immunotherapy

1. Introduction

Melanoma, a highly invasive and metastatictumor type, has presented increasing incidence andmortality in recent years [1–8]. The prognosis ofmalignant melanoma is related to the stage it is

0304-3835/$ - see front matter � 2008 Elsevier Ireland Ltd. All rightsdoi:10.1016/j.canlet.2007.12.017

* Corresponding author. Address: Disciplina de Imunologia,Universidade Federal de Sao Paulo, Rua Botucatu 862, 4� andar,Sao Paulo, SP 04023-901, Brazil. Tel.: +55 11 55496073; fax: +5511 55723328.

E-mail address: [email protected] (J.D. Lopes).

detected. In the majority of cases, early stages ofmelanoma are successfully treated with adequatesurgery, whereas unresectable or advanced meta-static disease is frequently refractory to conventionalchemotherapy. Although therapeutic strategies fordisseminated melanoma include chemotherapy,biochemotherapy, immune adjuvants, cytokines,vaccines, immunostimulants and monoclonal anti-bodies, only high-dose IFN-a and IL-2 treatments,that benefit a minority of patients, have beenapproved by FDA [9]. In this context, the develop-ment of novel therapeutic approaches as well as the

reserved.

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P.F.P. Mendes et al. / Cancer Letters 262 (2008) 276–285 277

finding of as yet non-existent reliable markers fordiagnosis or prognosis that can be used in clinicalpractice has been considered a promising area.

Melanoma metastatic potential may be associ-ated with molecules presented on the cell surfacewhose expression levels contribute to distinct meta-static phenotypes [10–12]. Although melanoma pro-gression is well known concerning its biological andclinical aspects, molecular mechanisms and markersassociated to metastatic dissemination still need tobe defined. Several oncogenes and tumor suppres-sors, as RAS, TP53, PTEN and CDKN2A, havebeen shown to be involved in melanoma pathogen-esis, but they have not been validated as prognosticmarkers. Monoclonal antibodies (mAbs) are one ofthe approaches used to identify such molecules cor-related with stepwise changes from normal skinmelanocyte to melanoma [13–16]. Monoclonal anti-bodies have enabled both the identification andmanipulation of these molecules, providing animportant new class of potential therapeutic agents.Their mechanisms of action include both directtumor cell destruction as indirect targeting ofgrowth and pro-angiogenic mediators [17]. Variousstudies have described mAbs against known mela-noma antigens that could be used as markers fordiagnosis and prognosis or as targets for immuno-therapeutic approaches [18–27]. However, no anti-tumor antibody is currently available for clinicaluse in melanoma [28] and markers defining differentphenotypes typical of unique populations of mela-noma are also not available.

Experimental models representing differentphases of melanoma carcinogenesis are still rare.In this context, B16 murine melanoma cells havecurrently been used as a model for studying tumorprogression due to their diversity, bringing insightsinto the metastatic process since it allows the isola-tion of subpopulations with different metastaticcapacities [29–32]. Previously, B16 melanoma vari-ants presenting different metastatic potential, as wellas in vitro growth kinetics, were obtained by in vitro

cloning and in vivo selection [33]. These subpopula-tions express different patterns of proteins and othercell surface components. In parallel, Oba-Shinjoet al. [34] described a novel melanocyte malignanttransformation model, where cell lines correspond-ing to different stages of melanoma genesis wereobtained after submitting a non-tumorigenic mela-nocyte lineage (melan-a) to sequential cycles ofanchorage blockade. This model includes non-tumorigenic cell lines corresponding to intermediate

phases of neoplastic transformation (3C and 4Ccells) and different melanoma cell lines (4C3�,4C3+, Tm1 and Tm5). In this way, both modelscan be used for the identification of moleculesexpressed specifically by these melanoma cells,which creates the perspective of studying tumordevelopment and heterogeneity and searching forhomologues in the human melanoma systems.

Here we report the production of an IgM mono-clonal antibody (G12F2) directed against B16 mela-noma cells that reacts with a Mr 230,000 protein onmalignant melanoma cells, but does not recognizeantigens in non-tumorigenic cell lines such asmelan-a melanocytes or intermediate cell lines, suchas 3C and 4C melan-a-derived cell lines. In addition,G12F2-recognized epitope showed to be higherexpressed as more metastatic the melanoma cellsare. Furthermore, G12F2 mAb effectively inhibitedin vitro cell proliferation, enhanced complementactivity against tumor cells and also inhibitedin vivo tumor growth and lung metastasis formation.Therefore, this mAb may be considered appropriatefor the development of new therapeutic agents andthe identification of molecular markers for mela-noma progression.

2. Materials and methods

2.1. Animals

Six weeks old male C57BL/6 mice were obtained fromthe animal facilities of Federal University of Sao Paulo(UNIFESP), Brazil. Male athymic Balb/C nude mice ofthe same age were purchased from the University of SaoPaulo, Brazil. The animals were maintained in a pathogenfree environment, with free access to food and water inaccordance with NIH Guide for Care and Use of Labora-tory Animals. All experiments were approved by theResearch Ethical Committee from UNIFESP.

2.2. Cell culture

Melan-a murine melanocyte lineage was cultured inRPMI, pH 6.9 (Sigma, St. Louis, MO, USA), containing5% fetal calf serum (FCS; Cultilab, Campinas, SP, Brazil)and 200 nM 12-o-tetradecanoyl phorbol 13-acetate(PMA; Tocris, MO, USA). Both non-tumorigenic (3C,4C) and tumorigenic (4C3�, 4C3+, Tm1 and Tm5) celllines derived from melan-a cells after sequential cycles ofanchorage blockade were cultured as melan-a cells, exceptfor PMA [34,35]. Murine fibroblasts (NIH 3T3), B16 mur-ine melanoma, B16 high and low metastatic variants andmel85 human melanoma cell lines were cultured as mono-

278 P.F.P. Mendes et al. / Cancer Letters 262 (2008) 276–285

layers in RPMI 1640 medium, pH 7.4, with 10% FCS. Allcell lines were maintained in 5% CO2 and 95% air humidatmosphere at 37 �C.

2.3. Immunization and production of monoclonal antibodies

Six weeks old male C57BL/6 mice were i.p. immunizedwith 106 B16 melanoma cells in Complete Freund’s Adju-vant (GibcoBRL, Grand Island, NY, USA). Mice wereboosted twice at two week intervals and three days afterthe last injection spleen cells were isolated and fused withp3.653 murine myeloma cells according to Lopes andAlves [36]. Cells from positive cultures were cloned by lim-iting dilution and expanded for ascitic fluid production byinjection of 107 hybridoma cells into the peritoneum cav-ity of mineral oil-primed athymic Balb/C mice. Class andsubclass of monoclonal antibody were determined usingthe Mouse Antibody Isotyping kit (BD Biosciences, SanDiego, CA, USA) following the manufacturer’s instruc-tions. For biological assays, G12F2 and an irrelevantIgM ascitic fluid were precipitated with 70% saturatedammonium sulfate.

2.4. Screening of monoclonal antibodies by whole cell

ELISA

For screening, B16 cells in FCS-free medium wereadded (2 � 104/0.1 mL/well) to 96-well tissue cultureplates and allowed to attach for 12 h in a humidifiedatmosphere of 5% CO2 at 37 �C. Cells were fixed with0.5% glutaraldehyde in PBS and blocked with 0.1% gly-cine in PBS. After, peroxidase endogenous activity wasblocked with 3% hydrogen peroxide in PBS. Wells wereblocked with 5% fat-free milk in PBS and hybridomasupernatants were added to the wells. After incubation,peroxidase-labeled goat anti-mouse IgM (Zimed, SanFrancisco, CA, USA) was added. Plates were washedand the substrate (1.0 mg of o-phenylenediamine in5.0 mL of 0.1 M citrate–phosphate buffer [pH 5.0] plus10 lL of 30% H2O2) was added. The reaction was inter-rupted with 4 N H2SO4. Optical density at 492 nm wasmeasured in an automatic MCC/40 plate reader (Labsys-tem Multiscan Dynatech, Chantilly, VA, USA).

2.5. Western blotting

Cells were lysed in NP-40 lysis buffer, containing a pro-tease inhibitor mixture (Sigma) according to the manufac-turer’s instructions, and protein concentration wasdetermined using the Bradford reagent (Bio-Rad, Hercu-les, CA). Equal amounts of protein (80 mg of total pro-tein) from each sample were resolved by reduced 7.5%sodium dodecyl sulfate–polyacrylamide gel electrophore-sis (SDS–PAGE) gels and blotted onto polyvinylidinefluoride membranes (Amersham, Piscatway, NJ). Non-specific sites were blocked by 5% free milk in PBS and

membranes were incubated with G12F2 hybridoma super-natants or diluted ascitic fluid (1:100) for one hour atroom temperature (RT). Bound antibodies were detectedwith peroxidase-labeled goat anti-IgM antibody for 1 hfollowed by developing using ECL detection reagent(GE Healthcare, UK). Total ERK expression (SantaCruz, CA) was used as internal control.

2.6. FACScan analysis

The binding of G12F2 mAb to cell surfaces was analysedby flow cytometry. After washing, 3T3, melan-a, 3C, 4C,Tm5 and B16 (106 cells) lineages were incubated withG12F2 or irrelevant IgM ascitic fluid (1:3 dilution) for 1 hat 4 �C. Cells were incubated with phycoerythrin (PE) orfluorescein isothiocyanate (FITC)-conjugated anti-murineIgM (Sigma) for 1 h at 4 �C protected from light, fixed in1% paraformaldehyde (Merck, Darmstadt, Germany) inPBS and examined by CellQUEST software using FACS-Callibur (Becton Dickinson Immunocytometry Systems).

2.7. Confocal microscopy

B16 cells (5 � 104) were cultured on glass coverslips inRPMI 1640 containing 10% FCS for 48 h. Cells were fixedin cold methanol and incubated with G12F2 or irrelevantIgM ascitic fluid (1:8 dilution) for 1 h. After this period,cells were incubated with fluorescein isothiocyanate(FITC)-conjugated anti-murine IgM (Sigma) with 50 lMDAPI (40,60-diamidino-2-phenylindole; Sigma) for nucleistaining plus 0.01% saponin (Sigma) in PBS-0.5% milkfor 1 h at RT, light protected, and sealed on glass slides.Images of stained cells were analysed in a Bio-Rad 1024UV confocal system attached to a Zeiss Axiovert 100microscope, using a 40� numerical-aperture 1.2 planapo-chromatic differential interference contrast water immer-sion objective.

2.8. In vitro proliferation assay

Viable 2 � 103 cells were added per well of 96-well platesand incubated for 24 h at 37 �C. After incubation, G12F2or control mAb were added in different concentrations(1.0 and 5.0 lg/mL) in complete medium. In order to mea-sure the daily proliferation rate, MTT [3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma] (0.5mg/mL) were added and incubated for 90 min at 37 �C[37]. Medium was discarded and 100 lL of cool isopropanolwas added for spectrophotometry reading at 570 nm.

2.9. Cytolytic activity mediated by complement

B16 cells (104 cells/well) were cultivated in 96-well platesduring 24 h at 37 �C. Next, adherent cells were incubatedwith guinea-pig complement (Gibco) according to the man-ufacturer’s instructions and G12F2 or control IgM at 0.5,


1.0, 2.5 and 5.0 lg/mL concentrations for 24 h and cell via-bility was measured by MTT assay. Cells incubated onlywith complement or mAb were used as controls.

2.10. Inhibition of tumor growth

B16 cells in exponential growth were harvested byexposure to 2 mM EDTA in PBS. Only viable cells(2 � 105), pre-incubated with 100 lg G12F2 or irrelevantIgM or PBS only, were used for injection in the scapularregion. C57Bl/6 mice (n = 5) were treated intravenouslywith G12F2, irrelevant IgM or PBS 2 days before tumorinjection and once weekly for 4 weeks. Growth of subcu-taneous tumors was measured with calipers at 2 day inter-vals. Tumor volume was determined as follows: d2 � D/2,where d and D represent, respectively, minor and majortumor diameters.

2.11. Experimental metastasis

To check the effect of G12F2 mAb on the number of B16melanoma lung nodules, 105 cells plus G12F2 or irrelevantIgM in PBS were intravenously injected into C57Bl/6 mice(n = 5). Intravenous treatment with mAbs (100 lg/mL)was made once a week for 3 weeks. Mice were sacrificed23 days after injection and lung metastatic coloniescounted.

2.12. Statistics

Analysis of variance and the Tukey–Kramer test wereused for statistical comparisons. All values were reportedas means ± standard error of the mean. p < 0.05 was con-sidered significantly different.

3. Results

3.1. The monoclonal antibody G12F2 recognizes a Mr

230,000 antigen on B16 melanoma cell surface

The mAb G12F2 was generated by immunization ofmale C57BL/6 mice with murine melanoma cell lineB16F10 associated with complete Freund’s adjuvant.The obtained G12F2 hybridoma produced an IgMk

immunoglobulin that recognizes a single sharp proteinband of approximately 230 kDa, as shown by Westernblot analysis of B16 total protein extract using G12F2mAb from peritoneal ascitis (Fig. 1A, left panel). Westernblot analysis using purified fibronectin as antigen dis-carded the possibility of G12F2 mAb is recognizing thishigh molecular weight extracellular matrix component(Fig. 1A, right and left panels). Fig. 1B shows thatG12F2 mAb, both ascitis and polyclonal serum, recog-nizes B16 intact cells by flow cytometry, with 99% and59% of positivity, respectively, compared to control IgM(only 20% of positivity). In addition, also considering that

animals were immunized with intact B16 melanoma cells,the cellular distribution of G12F2 antigen was evaluatedby confocal microscopy analysis. As shown in Fig. 1C,G12F2 ascitis reacts predominantly with B16 melanomacell membrane. No labeling was detected when cells wereincubated with the same concentration of an irrelevantIgM ascitis. These results indicate that the antigen recog-nized by G12F2 mAb is expressed on B16 cell membrane.

3.2. Mr 230,000 antigen is not expressed in non-tumorigenic

cell lines and correlates with metastatic capacity

To evaluate the especificity of G12F2 mAb, the pres-ence of G12F2-recognized antigen was analysed in othercell lines, both tumorigenic and non-tumorigenic. Proteinextracts from the non-tumorigenic cell lines melan-a mel-anocytes (ma; C57BL/6 background) and 3T3 fibroblasts(Balb/C background) were not detected by G12F2 mAbby Western blot (Fig. 2A and data not shown, respec-tively). Moreover, Mr 230,000 molecule was recognizedby G12F2 on 4C3+, Tm1, Tm5 and B16 tumorigenic lin-eages, but not on non-tumorigenic 4C cell line, whichwas derived from melan-a cells and corresponds to anintermediate stage of melanocyte malignant transforma-tion (Fig. 2A) [34,35]. Interestingly, G12F2 antigenshowed to be highly expressed in more aggressive mela-noma cells, like more metastatic B16 lineage (B16+)compared to less metastatic one (B16�). All 4C3�,4C3+, Tm1 and Tm5 are tumorigenic in vivo, but havedifferent latency time for tumor appearance. Differentfrom the others that result in tumors growing fast,4C3� presents slow tumor growth rate, both in vitro

and in vivo [44]. Although 4C3�, 4C3+, Tm1 and Tm5melanoma cells are able to result in tumor formation,the ability to form lung metastatic colonies was not stillanalysed in these cells. Comparative study of metastaticpotential among the different melanoma cell lines areunder investigation in our laboratory.

Previously, we described isolation of variants from B16cell line with different in vivo metastatic patterns andin vitro growth kinetics [33]. Mr 230,000 could be a differen-tial marker of B16 subpopulations, since Western blottinganalysis showed that this molecule was expressed inincreased levels on high metastatic B16 compared to lowmetastatic B16 extracts (Fig. 2A), suggesting that this anti-gen could be associated with B16 metastatic phenotype.

G12F2 antigen cell surface expression was also evalu-ated in melan-a melanocytes, intermediate cell lines 3Cand 4C cells, and in Tm5 melanoma lineage by flowcytometry using G12F2 ascitis. This data showed thatthe molecule recognized by G12F2 mAb is not expressedin both melan-a and intermediate cell lines 3C and 4C, butis significantly expressed on Tm5 melanoma cell surface(47% of positivity; Fig. 2B). Taken together, these resultssuggest that Mr 230,000 molecule is a metastasis-associ-ated tumor-specific antigen.

Fig. 1. G12F2 monoclonal antibody recognizes a 230 kDa molecule on B16 melanoma cell surface. (A) As shown by Western blotanalysis, G12F2 mAb peritoneal ascitis recognizes a single sharp band presenting 230 kDa (left panel, lane B16) in B16 melanoma cellprotein extract, but does not recognize purified fibronectin (left panel, lane FN). The presence of fibronectin in B16 protein extract (rightpanel, B16 lane) and purified fibronectin fraction (right panel, lane FN) is shown by Western blot using a polyclonal serum againstfibronectin. The band corresponding to fibronectin does not have the same molecular weight of G12F2 antigen (left and right panels, B16lane). (B) G12F2 mAb, both from polyclonal serum or peritoneal ascitis, reacts with cell surface of viable B16 cells, as shown by flowcytometry. A control group represents B16 cells incubated with an irrelevant IgM monoclonal antibody. Dashed lines indicate cellsincubated in the absence of primary antibody and filled curves, experimental groups. Numbers inside the graphs represent percentage ofpositive cells compared to respective control group. (C) Confocal microscopy of B16 melanoma cells showing that the G12F2 epitope islocalized on cell surface. Left panels show phase contrast microscopy and right ones cells incubated with irrelevant IgM ascitic fluid (1:10dilution) or with G12F2 ascitic fluid (1:10 dilution) (green staining). DAPI staining was used to visualize nuclei (blue staining).


Fig. 2. G12F2 antigen is expressed by aggressive melanoma, but not by non-tumorigenic cells. (A) Protein extracts from non-tumorigenicmelan-a (ma) and 4C (corresponding to an intermediate phase of malignant transformation) melanocytes, and tumorigenic melanoma cells(4C3�, 4C3+, Tm1, Tm5, B16, B16� and B16+) were analysed by Western blot using G12F2 mAb (1:100 dilution). Mr 230,000 band wasobserved in all cell lines, except in non-tumorigenic melanocytes (melan-a and 4C) and in a non-aggressive melanoma lineage (4C3�).Note that G12F2 epitope is strongly expressed as more metastatic and aggressive melanoma cells are (4C3+ and B16+). ERK expressionwas used as an internal control for sample loading. The numbers below this figure indicate the G12F2/ERK ratio, obtained after bandsquantification using ImageJ software. (B) Non-tumorigenic melanocytes melan-a (ma), 3C and 4C lineages (corresponding to intermediatephases of malignant transformation), and Tm5 melanoma cells were analysed by Flow cytometry using G12F2 mAb ascitis (filled curves)or an irrelevant IgM ascitis (control group, dashed lines). Numbers inside the graphs indicate the percentage of G12F2-positive cellscompared to the respective control group.


3.3. G12F2 mAb inhibits both murine and human melanoma

cell proliferation and enhances complement-mediated

cytolytic activity against B16F10

Next, we investigated whether Mr 230,000 moleculeneutralization by G12F2 mAb could affect in vitro mel-anoma cell proliferation. G12F2 ascitis treatment inhib-ited in vitro B16 cell growth, both at 1.0 (Fig. 3A) and5.0 lg/mL (data not shown) concentration, as comparedwith the cells treated with the same concentration of anirrelevant IgM ascitis. This mAb was also able to signif-icantly reduce mel85 human melanoma cell proliferation(Fig. 3B), indicating that this mAb may exert its tumorinhibitory properties also in human tumor cells. BothB16 and mel85 melanoma cells showed clearly growth

arrest until the fourth day when cultivated in the pres-ence of G12F2 mAb. This effect was statistically signif-icant on the fourth day, after which growth resumed.The finding that no cell detachment was observed aftertreatment with G12F2 mAb points to the possibilitythat the target antigen does not seem to be a adhesionmolecule. Because B16 cells are susceptible to comple-ment lysis, we evaluated the in vitro G12F2 mAb cyto-toxicity in the presence of complement. An increasedcytotoxic effect against B16 cells was observed in thepresence of increasing G12F2 mAb concentrations. Bothaddition of complement and mAb alone slightly reducedcell viability, but this cytotoxic activity was significantlyincreased in the presence of both, complement and mAb(Fig. 3C).

Fig. 3. G12F2 mAb inhibits both murine and human melanomacell proliferation and increases cytotoxicity by complement againsttumor cells. Adhered murine B16 (A) and human mel85 (B)melanoma cells (104 cells/well) were incubated for 24 h with G12F2(G12F2 mAb), control IgM mAb (Ctrl mAb) or only medium(medium). The G12F2 mAb ascitis concentration used was 5.0 lg/mL for B16 cells and 1.0 lg/mL for mel85 cells. Cell proliferationrates were determined during 5 days by MTT assay. These resultswere done in triplicates and are representative of three-independentexperiments. (C) B16 (104 cells/well) were incubated for 24 h withG12F2 (1.0, 2.5 and 5.0 lg/mL), in the presence or absence ofguinea pig complement 1:80 diluted (compl). At the end ofincubation period, viable cells were measured with MTT. Controlswere done with cells incubated only with complement or G12F2and cells incubated in the absence of both mAb and complement.*p < 0.05, **p < 0.001.


3.4. G12F2 mAb inhibits in vivo tumor growth and

metastasis

The capacity of G12F2 mAb to affect in vivo tumorestablishment in C57Bl/6 mice was investigated. Rapidtumor growth was observed in mice treated with bothirrelevant IgM or PBS (Fig. 4A), while growth was signif-icantly arrested in G12F2-treated mice, suggesting thatthis mAb was protective in this model. In addition,G12F2 mAb anti-metastatic potential was evaluated byexperimental lung metastasis assay in C57BL/6 miceintravenously treated with this mAb. As shown inFig. 4B, the number of metastatic nodules was signifi-cantly reduced in G12F2-treated compared to irrelevantIgM- or PBS-treated mice, with an average nodule num-ber of 26 ± 2.8, 93 ± 10 and 106 ± 11, respectively. Also,G12F2-treated mice developed nodules smaller in sizethan those seen in the control groups (Fig. 4B). Theseresults show that treatment with G12F2 mAb decreasedmetastasis development by B16 cells, suggesting theinvolvement of G12F2 antigen in metastatic process. Fur-ther experiments are already being done in our laboratoryto determine whether G12F2 mAb presents inhibitoryactivity against established tumors, thus becoming apotential therapeutic tool.

4. Discussion

Malignant melanoma is the most aggressive formand causes the majority of deaths from skin cancers.The prognosis of malignant melanoma is stronglyrelated to the stage at which it is detected. Earlystages of melanoma are, in majority of the cases,successfully treated with surgery [38], but advanceddisease is generally refractory to conventional che-motherapy. The disappointing results that havecome from chemotherapy show 5-year survival ratesfor treated patients ranging from only 3% to 14%[28,39]. For these reasons, the appropriate systemictreatment for disseminated malignant melanoma isstill controversial and no defined standard exists.Therefore, there is much interest in the developmentof novel therapeutics for this kind of cancer, spe-cially immunotherapy and targeted molecules.Among therapeutic strategies for metastatic mela-noma, the only immunological approach approvedby FDA in the last 30 years has been high-dose ofIFN-a and IL-2. However, both agents are highlyexpensive, present considerable toxicity and only aminority of patients benefit from this treatment interms of long-term survival [40]. Although mono-clonal antibodies have had significant impact oncurrent practice in oncology [17,41,42] and mela-noma has been considered susceptible to immuno-

Fig. 4. G12F2 mAb reduces significantly both in vivo tumorgrowth and lung metastasis formation. (A) Six weeks old C57Bl/6male mice (n = 5), previously treated with G12F2 mAb, irrelevantIgM mAb (control mAb) or PBS, were injected with 2 � 105 B16cells. Treatment was performed during three weeks after tumorcells implantation. Minor (d) and major (D) diameters of tumorswere determined using calipers each 2 days. Tumor volumes weredetermined as follows: d2 � D/2. The data represent the meanvolume observed in five animals per group. (B) B16 cells (105) wereintravenously injected into C57Bl/6 mice and treated with G12F2,irrelevant IgM (Ctrl mAb) mAb or PBS as described in Section 2.After 21 days, animals were sacrificed by cervical dislocation, andthe number of lung metastatic colonies determined. ***p < 0.0005.(C) Lungs from C57Bl/6 mice bearing melanoma, treated withPBS, control IgM (Ctrl mAb) or G12F2 mAb.


therapy, no antitumor antibody against melanomais currently available for clinical use [9,43].

Herein we describe a mAb, G12F2, recognizing ahigh molecular weight antigen of approximately230 kDa, present on the cell surface of murine mel-

anoma but not in non-tumorigenic cells (Figs. 1 and2), that has the capability of inhibiting proliferation(Fig. 3A and B) and in vivo tumor growth and lungmetastases formation (Fig. 4), and increases com-plement-mediated cytolytic activity (Fig. 3C). Moremalignant melanoma cells, as seen by the number oflung colonies, showed higher expression of this mol-ecule compared with less malignant cells, suggestingthat the antigen recognized by G12F2 mAb could befunctionally relevant only for tumorigenic cells, spe-cially metastatic cells.

Several investigations suggested that changes insurface molecules could be important in cancermetastasis [31]. The fact that mAbs can only acton molecules expressed on the cell surface orsecreted makes the cell-surface expressed Mr

230,000 antigen a promising target for carcinogene-sis and metastasis studies in the murine melanomamodel (Fig. 1). In addition, considering the inhibi-tory property presented by G12F2 mAb onin vitro human tumor growth (Fig. 3B), the possibil-ity of human melanoma cells expressing a homo-logue molecule assumes great importance and nowis under investigation.

The ideal target antigen should be expressed as atumor-specific molecule [18]. In this way, G12F2mAb did not bind and/or affect normal cells as mel-anocytes and fibroblasts (Fig. 2A and data notshown) and instead recognized only tumorigenicmelanoma cells such as 4C3+, Tm1, Tm5 andB16F10. On the other hand, the non-tumorigenicmelan-a-derived cell lines, 3C and 4C, correspond-ing to early phases of melanocyte malignant trans-formation, were not recognized by G12F2 mAb(Fig. 2), suggesting that Mr 230,000 molecule mayrepresent a novel highly malignant murine mela-noma marker. Phenotypic markers that discriminatemelanocytes from melanoma can be particularlyuseful for stage differentiation in melanoma pro-gression [30]. Western blotting analysis demon-strated that the 230 kDa molecule has itsexpression increased on B16 high metastatic variantextract when compared with low metastatic variant(Fig. 2A), thus making the Mr 230,000 molecule adifferential antigen among subpopulations.

The major characteristic of malignant cells istheir ability to invade tissues and form metastaticfoci at distant locations in the host, a process whichrequires cellular proliferation. G12F2 mAb affected,but not completely abolish, in vitro growth tumorcell proliferation, since B16 cells resumed prolifera-tion four days after treatment (Fig. 3). This result


allows the speculation that the target antigen ofG12F2 mAb could be a chemokine or growth factorreceptor. The anti-proliferative activity of G12F2was confirmed in vivo, since tumor growth inG12F2-treated mice was reduced during the treat-ment but was restarted after its interruption, whileIgM or PBS control treatments did not affect tumorgrowth (Fig. 4A). This type of protection, appar-ently mediated by antibodies, has not yet beenreported, thus conferring even more importance toour results. Also, they allow to assume the possibil-ity of the use of our mAb as an addition to conven-tional therapy. Since most tumors, in special solidtumors, are multifactorial and are frequently linkedto defects in more than one signalling pathway,multi-targeting therapy might be more efficient,not only to eliminate tumor cells, but to limit theappearance of drug resistance.

Therapeutic mAbs may trigger different immune-effector mechanisms, such as antibody-dependentcellular toxicity, complement-dependent cytotoxic-ity and complement-dependent cell-mediated cyto-toxicity [41]. Inhibition of B16 growth by G12F2mAb could have resulted from complement activa-tion, since its addition to B16 culture plus comple-ment significantly increased cell lysis (Fig. 4C).However, other mechanisms, that could actin vivo, cannot be ruled out as yet.

Malignant cells are not inherently metastatic butprobably develop this capacity during tumor pro-gression as they acquire or express new characteris-tics through a process of selection fromcontinuously arising variants [28,31]. G12F2 MAbsuppressed lung metastasis of B16 cells in mAb-trea-ted mice. Three weeks after injection, control groupsdeveloped a greater number of lung colonies withbigger diameter as compared to G12F2-treatedgroup, which presented less and smaller lung meta-static colonies (Fig. 4). According to our data,G12F2 antigen expression correlated with the meta-static phenotype of murine melanoma and, besidesaligning with markers of prognosis, opens otherpossibilities of becoming a novel target for immuno-diagnostic approaches and must lead to furtherinvestigations to determine whether it has anequally relevant human homologue, since G12F2mAb also significantly inhibited human melanomagrowth in vitro. In this way, G12F2 mAb may behelpful to better understand some crucial eventsalong the metastatic cascade that characterize both,this melanoma model as well as the human cancer.It also may represent novel melanoma target espe-

cially attractive in the development of new adjuvantagents that can be more effective to treat this aggres-sive disease [42,43].

Acknowledgement

This work was supported by Fundac�ao deAmparo a Pesquisa do Estado de Sao Paulo(FAPESP).

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A cell surface 230 kDa protein from murine melanoma involved with tumor malignancy

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