[CANCER RESEARCH 48,4954-4958, September 1, 1988]

Expression and Modulation of a Retrovirus-associated Antigen by Murine

Melanoma CellsStanley P. L. Leong,1Jacqueline Muller, Robert A. Verter, ElìeserGorelik, Tsuyoshi Takami, and Vincent J. Hearing

Department of Surgery and Arizona Cancer Center, University of Arizona Health Sciences Center, Tucson, Arizona S5724 [S. P. L. L.]; Division of Biochemistry andBiophysics, Food and Drug Administration Center for Drugs and Biologies, Bethesda, Maryland 20892 [J. M.J; Research Service, Veterans Administration MedicalCenter, Baltimore, Maryland 21218 [R. A. Y.J; Department of Pathology, Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213 [E. G.J;Department of Pathology,Section 2, Gifu University School of Medicine, Gifu-City, 500, Japan [T. T.J; and Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland 20892¡V.J. HJ

ABSTRACT

The antigen recognized by the monoclonal antibody MM2-9B6 isspecific for melanomas originating in CS7BL/6 mice. It is expressed bythree melanomas of independent origin and not by normal or fetal tissues,by a wide variety of other nonmelanoma tumors in CS7BL/6 mice, oreven by melanomas syngeneic to other strains of mice. We have demonstrated that the expression of the relevant antigen is dependent on thereplication and budding of a B-tropic ecotropic murine retrovirus. Therelationship between the expression of this virus and carcinogenic progression may yield important insights into the cascade of events leadingto neoplastic transformation.

INTRODUCTION

Since its isolation more than 3 decades ago from a spontaneous neoplasm in C57BL/6 mice, the B16 malignant melanoma has been extensively studied as a model for neoplastictransformation, tumor heterogeneity, invasion, and metastasis.More recently, the B16 melanoma has served as a convenientprototype for studies on the immunological management ofprogressive tumor growth and metastasis, and a plethora ofmonoclonal antibodies of varying specificities have been raisedtowards this end (1-7). Perhaps the most specific of theseantibody probes are MM2-9B6 (an IgG2b) and MM2-3C6 (anIgM) (8,9). The murine hybridomas producing these antibodieswere obtained from syngeneic C57BL/6 mice immunized withB16 cells. These antibodies apparently recognize the same, asyet undefined, epitope, and are exquisitely monospecific for theB16 cell line (8, 9). In the present studies we have determinedthat the relevant antigen is related to the expression of a murineretrovirus which can be isolated from melanomas in C57BL/6hosts.

In previously published work, the antigen recognized byMM2-9B6 and MM2-3C6 was found to be expressed by all 3sublines of the B16 melanoma examined, as well as in 50different clones of the B16 melanoma that were generated, butwas not detectable on the Cloudman S91 or Harding-Passeymurine melanomas, on any of 11 other murine nonmelanomatumors, or on 8 syngeneic normal murine adult or fetal tissues.The antigen was also absent from 6 human melanomas examined (8, 9). Flow cytometric analysis using dual parameterstaining (with fluorescein-labeled antibodies to detect antigenand propidium iodide staining to measure DNA content) revealed that expression of the antigen on the cell surface wasassociated with cell proliferation; more than 90% of the cellswere antigen positive during active growth, but there was asignificant decrease in antigen expression after several days inthe stationary phase (all antigen-negative cells were in the Go-Gi phase of the cell cycle). In this study we have further

Received 2/29/88; revised 5/26/88; accepted 6/3/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.

examined the antigen specificity in more recently derived murine melanoma cell lines and have characterized the nature ofits expression. We have found that this antigen is expressed bytwo new chemically induced murine melanomas of C57BL mice(in addition to the B16), and that the production of the antigenis related to the presence and budding of a B-tropic ecotropicmurine retrovirus.

MATERIALS AND METHODS

Cells and Tissue Culture. The B16 Fl, B16 FIO, and B16 FLRmelanoma cell lines were gifts from Dr. Artemio Ovejera; the B16 BL6,B16 013, and B16 Bl Sb melanoma cell lines were gifts from Dr. GarthNicolson; other murine melanoma lines and other tumor lines wereobtained as previously noted (10-12). Cells were routinely grown toconfluence (when antigen expression is maximal) in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum and

antibiotics as previously detailed (12,13). The cells were harvested witha brief (30 s) trypsin treatment and washed twice with Hanks' balanced

salt solution prior to use.Flow Cytometry. Cells (IO6) were incubated in 12- x 75-mm tubes

with 50 ¿ilof MM2-9B6 or MM2-3C6 culture supernatant for 30 minat 4°C,as previously described (8); the cells were then washed twice

and incubated with a fluorescein isothiocyanate-conjugated secondaryantibody (goat IgG anti-mouse immunoglobulin) for 30 min at 4*C,

washed twice again, and quantitated in a Cytofluorograf 30-11 systemwith a Model 2150 computer. Both antibodies gave virtually identicalresults, and only the data from MM2-9B6 are presented in the tablesand figures. Backgrounds were established in this and the other immunological assays either by omitting the first antibody, or by using anirrelevant MOPC 141 (IgG2b) monoclonal antibody.

Enzyme-linked Immunosorbent Assay. Cells (IO6)were incubated with50 ill of MM2-9B6 as above, but the secondary antibody used was aperoxidase-conjugated secondary antibody (rabbit IgG anti-mouse immunoglobulin), and specific binding was quantitated, after thoroughsubsequent washing, by reactivity with orthophenylenediamine substrate, as detailed previously (4).

Electron Microscopy. Cells (5 x IO6) were incubated with 250 ^1 ofMM2-9B6 antibody as above, but the secondary antibody used wascolloidal gold-conjugated (rabbit anti-mouse IgG); after thorough washing, the cells were fixed in glutaraldehyde, embedded in Epoxy resin,thin sectioned, and stained with lead citrate and uranyl acetate in theusual manner. The sections were viewed and photographed using aHitachi H500 electron microscope.

Infections with Virus. SC-1 fibroblast cells were infected with theecotropic viruses noted in Table 2, at a low multiplicity of infection inthe presence of polybrene (8 Mg/ml); the infected cells were then assayedfor expression of virus and antigen by immunoelectron microscopy andELISA,2 as noted above.

RESULTS AND DISCUSSION

In these studies we have extended our specificity analysis andexamined the expression of the antigen recognized by MM2-9B6 and MM2-3C6 on additional sublines of the B16 mela-

2The abbreviations used are: ELISA, enzyme-linked immunosorbent assay;

DMBA, dimethylbenzanthracene; MNNG, methylnitronitrosoguanidine.

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MELANOMA RETROVIRUS-ASSOCIATED ANTIGEN

Table 1 Reactivity 0/MM2-9B6 monoclonal antibody with murine tumor linesCell lines as noted were grown to confluence in Dulbecco's modified Eagle's medium, harvested, and washed twice prior to use. Visible pigment was estimated in

the cell pellets on a scale from white (—)to black (++++). Analysis by flow cytometry, the ELISA assay, and immunoelectron microscopy was performed as detailedin "Materials and Methods."

CelllineMelanomasB16FIB16F10B16FLRB16BL6B16O13B16B15bB16T2B16C39JB/RHJB/MSK1735S91Harding-PasseyOther

syngeneic celllines112fibrosarcomaT92497sarcomaGL26gliomaFBL3leukemiaRBLS

leukemiaDescriptionLow

metastaticHighmetastaticLymphocyteresistantHighinvasiveOvarianmetastaticBrainmetastaticMNNGtreatedMNNGtreatedDMBAinducedDMBAinducedUVinducedSpontaneousSpontaneousUV

inducedMethylcholanthreneinducedSpontaneousVirus

inducedVirusinducedStrain

oforigin(réf.)C57BL/6

(28)C57BL/6(28)CS7BL/6(29)C57BL/6(30)C57BL/6(31)C57BL/6(32)C57BL/6(27)C57BL/6(27)C57BL/600)C57BL/600)C3H

(33)DBA(34)Wild-type(35)C57BL/6

(36)C57BL/6(37)C57BL/6(38)C57BL/6(39)C57BL/6

(39)Flow

Visible cytometrypigment(%)+

92+++99+

96++++9598+++

96BKGBKG97+++

88BKG+

BKG++BKGBKGBKGBKGBKGBKGElectron

microscopyImmunoglobulin-

ELISA goldstain+

NT"+

++NT+NT+NT+NT————++++——NT—

——

————

—NTNTVirus

buddingNT+NTNTNTNT——+++NT+———NTNT

' NT, not tested; BKG, background.

noma (Table 1). Our previous studies (8, 9) had shown that theantigen was expressed on the B16 Fl, the B16 FIO, and theB16 FLR sublines, and this was substantiated in the currentstudy. We have now found in addition that the antigen isexpressed on several other distinct B16 sublines which havebeen selected on the basis of their metastatic properties. Forthe B16 BL6, the B16 013, and the B16 B15b sublines, antigenwas expressed on more than 95% of the cells, as determined byflow cytometry. There was no correlation of antigen expressionwith the pigmentation of these lines. However, the antigen wasnot expressed by 2 B16 sublines (B16 T2 and B16 C39) thathad been treated with MNNG (see below). In other experimentswe have also determined that there is no correlation in theexpression of this antigen with the expression of 112 antigens(data not shown).

As noted previously (8,9), the S91 and Harding-Passey tumor

lines were negative for antigen expression as was the recentlyUV light-induced K1735 melanoma of C3H mice. Surprisinglyhowever, both the JB/RH and the JB/MS melanomas werefound to express high levels of this antigen (Table 1), the firstcell lines other than B16 melanoma to do so; again, there wasno correlation of antigen expression with pigmentation, as theJB/RH is amelanotic and the JB/MS is highly pigmented (10-12). It is interesting to note that both the JB/RH and the JB/MS tumors originated in C57BL/6 mice (as did the B16 tumor),although they were generated by the chemical carcinogenDMBA rather than by spontaneous events. We immediatelysought to determine if other nonmelanoma tumors of C57BL/6 origin (including a UV-induced fibrosarcoma, a chemicallyinduced sarcoma, a spontaneous glioma, and two virus-inducedleukemias) also expressed the antigen, but found that they didnot (Table 1); the antigen was undetectable by flow cytometry,by an ELISA, or by electron microscopic immunohistochemicalcriteria.

The expression of this melanoma-specific antigen is unusually stable, and it has shown no significant change in expressionover more than 50 sequential passages. Antigenic heterogeneityis one of the major problems facing tumor immunologists (14-16), and thus the relatively stable expression, as well as the

specificity, of the antigen identified by the MM2-9B6 andMM2-3C6 antibodies has made this an attractive model forradioimaging and immunotherapy studies. Very recently, infact, MM2-9B6 has been successfully used for immunotherapyagainst the metastatic spread of B16 melanoma cells (17). Theidentification and characterization of this antigen were therefore thought to be highly pertinent to the potential use of theseantibodies as diagnostic or therapeutic tools. Thus far, however,all attempts to identify and characterize the relevant antigenhave failed. Unsuccessful techniques that have been attemptedincluded surface iodination and immunoprecipitation, metabolic labeling with amino acid and carbohydrate precursorsfollowed by immunoprecipitation, and Western immunoblot-

ting of solubili/ed B16 cell extracts (Refs. 8 and 9; Footnote3).

Our concurrent studies using flow cytometry and immunoflu-orescence microscopy revealed that the surface expression ofthe antigen was quickly modulated upon reaction with theMM2-9B6 or MM2-3C6 antibodies. The distribution of theantigen on the surface of B16 cells was initially uniform, butwithin minutes following exposure of cells to the antibody at23°Cor 37°C,the antigenrantibody complexes appeared to form

a patch on the cells and became undetectable within 30 min,unless the incubation was performed at 4°Cin the presence of

azide (data not shown). In studies aimed at determining if theloss of the antigen:antibody complexes from the surfaces of theB16 cells reflected their intermi li/ut ion. or their sloughing offinto the extracellular milieu, we performed immunocytochem-istry at the electron microscopic level. Those studies (whichwere performed only with MM2-9B6, an IgG, for technical

reasons) revealed that some of the complexes were internalizedand could be seen in the cytoplasm within 15 min (Fig. l ). Theseanalyses also revealed that the bound antibody was often foundin close association with virus particles budding from elongatedcell processes. We initially discounted the possibility that avirus encoded the relevant antigen because of the extremelyrestricted specificity of the antigen, the lack of its expression

3 Unpublished results.

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Fig. 1. Expression of antigen by murine melanomas of C57BL/6 mice. Melanoma cells werereacted with MM2-9B6 and colloidal gold-conjugated second antibody and processed for electronmicroscopy as noted in "Materials and Methods."

The colloidal gold localization of antibody can beseen as regular, circular dark black dots; all controlcell populations, determined by substitution of anirrelevant antibody, were negative for antibodylocalization (not shown). Throughout these studies, we observed the same pattern of gold labelingin all positive samples; the IS-nm gold particleswere localized on cell processes and in varyingnumbers associated with viral particles. Since weused only one staining protocol, we do not yetknow whether this pattern reflects the true distribution of antigen or the fact that our methodresults in the visualization of only a portion of theantigen actually present. Top, low magnificationof B16 cells showing cellular processes, buddingvirus particles with associated antibody (encircled),and antibody associated with cell processes (arrows): x 22,000. Bottom left, B16 cells postincu-bated with antibody IS min at 37*C, showing

internalization of antigen:antibody complexes; /.,lysosome; M, melanosome; x 39,000. Bottomright, high magnification of B16 cells showingantibody association with virus particles and cellular processes; x 52,500.

on virus-transformed cells syngeneic to C57BL/6 mice (RBL5and FBL3), and because the antigen-positive cells had testednegative for the following murine viruses: polyoma virus; ad-enovirus; hepatitis virus; minute virus; lymphocytic choriom-eningitis virus; ectromelia virus; reovirus type 3; pneumoniavirus; K virus; Theiler's virus; Sendai virus; and lactate dehy-

drogenase virus.In order to definitively show that the MM2-9B6 antibody

recognizes a virus-induced or virus-encoded antigen, a cell-freesupernatant from B16 FIO cells was used to infect the SC-1murine fibroblast cell line. SC-1 cells, which were antigennegative and virus negative prior to infection (Fig. 2), nowshowed abundant virus production and specific binding of theantibody (Fig. 2), which clearly demonstrates the virus-associated nature of this antigen. Further analysis of the specificityof MM2-9B6 was performed by examining its reactivity withSC-1 cells infected with various types of known ecotropicviruses (Table 2). Infection of SC-1 cells with the virus isolatedfrom B16 cells resulted in expression of the antigen recognizedby MM2-9B6. Infection with B-tropic virus isolated fromC57BL/6 mice did not result in expression of MM2-9B6 reactive antigen, nor did infection with ecotropic murine retrovirusfrom AK.R mice or wild mice (Table 2). However, infection ofSC-1 cells with a B-tropic virus from BALB/c mice (WN1802B)or an N-tropic virus from C57BL/6 mice (1920) did result inexpression of the relevant antigen on the infected SC-1 cells(Table 2). Thus, while the antigen recognized by MM2-9B6 isnot uniformly assoicated with ecotropic virus infection, neither

is it solely associated with infection with the virion present inC57BL melanomas. This type of seemingly nonuniform reactivity with monoclonal antibodies against murine retroviruseshas been previously reported (18); the clarification of the reasons behind this pattern of reactivity will require identificationand characterization of the relevant antigen.

The existence of type C virus particles in murine melanomashas been known for some time (19-21), although a causativerelationship of the virus with melanoma has never been established. More recently, the presence of type C virus particles hasbeen shown to elicit immune responses in naive C57BL/6 hostsagainst B16 melanoma (22, 23), but the virus has not beenfurther identified. These studies have been difficult to assesssince most B16 sublines in use have been thought to be virusfree, at least with respect to those viruses that are routinelytested for (as listed above). Our results are consistent with theabove studies; our B16 sublines, and the JB/RH and JB/MSlines, have been tested, and found negative, for a variety oftypical murine viruses, as noted above, yet these cells areexpressing type C virus particles. It is further obvious that theB16 cells elicit an immune humoral response in syngeneic hostswhich is directed against a virus-associated antigen, and whichwas responsible for the initial generation of these two highlyspecific monoclonal antibodies. It is appropriate to note thatother types of immune responses against these various cell linesare also elicited which are directed against common melanoma-specific antigens, and which cross-react with murine melanomasother than those syngeneic to C57BL/6 hosts (10-13); those

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Table 2 Reactivity ofMM2-9B6 with virus-infected SC-1 cellsSC-1 fibroblast cells were infected with the ecotropic viruses noted and assayed

for expression of virus and antigen by immunoelectron microscopy and ELISA;the data are presented as detailed in Table 1.

--:

4!M*

, •k—.•j-•

Fig. 2. Expression of antigen in virus-infected SC-1 cells. SC-1 cells werereacted with MM2-9B6 or NS-I (as noted below) and stained for antibodylocalization as noted in Fig. 1. All control cell populations were negative forantigen expression, as were all other infected SC-1 cells listed in Table 2. Top,uninfected cells, reacted with MM2-9B6; x 12,300. Middle, cells infected withB16 FIO virus, reacted with NS-1; x 35,700. Bottom, cells infected with B16 FIOvirus, reacted with MM2-9B6; x 41,000.

antigens, which are not virus related or virus associated, arethus distinct from the antigen studied in this report.

An interesting observation was the lack of antigen expressionon the two sublines of B16 cells that had been treated withMNNG (B16 T2 and B16 C39). When those cells were examined by immunoelectron microscopy, none of the cells examinedhad virus budding at the cell surface. This suggests that expression of the antigen is related to the replication and budding ofvirus particles. It is possible that the B16 T2 and B16 C39sublines may contain antigen within the cells, but are negativeby our criteria because the antigen is not expressed at the cell

EcotropicinfectingvirusNoneB16F10LP-BM51909WN1802B2950Moloney1920AKV623Cas-2-SType

or origin ofvirusB-tropic

from B16melanomaB-tropicfrom C57BL/6miceB-tropicfrom C57BL/6miceB-tropicfrom BALB/cmiceB-tropicfrom BIO.FashenNB-tropic

virusN-tropicfrom C57BL/6miceN-tropicfrom AKRmiceN-tropicfrom wild miceElectron

microscopyI

ninninoVirusglobulin-budding

goldstain__+++—+—+++—-i--+++—+ELISA_+NT*—+NT-——-

" NT, not tested.

surface. The alteration of cell surface expression of membraneproteins and/or antigens by treatment with MNNG has beenpreviously documented (24-27).

In summary, we have shown that the antigen recognized bythe MM2-9B6 and MM2-3C6 antibodies is expressed not only

by the spontaneously derived B16 melanoma, but also by 2 newchemically induced melanomas. We have determined that antigen expression is related to the presence and budding of a B-tropic ecotropic murine retrovirus. Of special interest is themechanism whereby this virus-related antigen is expressed bymurine melanoma cells in such a highly specific nature. Wehave shown that the relevant antigen is produced by 3 of 3melanomas of C57BL/6 origin, but by 0 of 5 nonmelanomaneoplasms of C57BL/6 origin. The relationship between theexpression of this virus and the events which led to the transformation of the melanocytes remains a matter of conjecture atthis point. The explanation of a trivial laboratory contamination error has been tentatively ruled out because the virus isexpressed in virtually all B16 sublines examined as well as inthe recently derived JB/RH and JB/MS melanomas (whichwere generated at and obtained from many independent research institutions). The virus could have been incorporatedinto the genome as the result of tumor passage in mice; however,this also seems improbable considering the past history of allthe different sublines of B16, and for the more recently generated JB/RH and JB/MS melanomas. In addition, the Harding-Passey tumor which originated in wild mice has been passagedfor more than 1 yr in our laboratory in BALB/c mice, andalthough type C virus particles are present in these cells, andare actively budding, this melanoma cell line has not becomeantigen positive. The most likely explanation consistent withthese results, in our opinion, is that the virus present in transformed melanocytes is a recombinant form of the endogenous,ecotropic virus of C57BL/6 mice. Studies to further identifythis virus, and the mechanism by which it is specifically expressed in transformed melanocytes, are currently under way.The elucidation of the processes involved in the expression ofthis virus and their possible relationship to the neoplastictransformation of these cells are an exciting subject for futurestudy.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Albert DeLeo, Dr. DouglasGersten, Dr. Lloyd Law, and Dr. Richard Niles for their helpfuldiscussions and critical reviews of the manuscript.

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1988;48:4954-4958. Cancer Res   Stanley P. L. Leong, Jacqueline Muller, Robert A. Yetter, et al.   by Murine Melanoma CellsExpression and Modulation of a Retrovirus-associated Antigen

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