[CANCER RESEARCH 35, 2520-2531, September 1975]

SUMMARY

Tumor-associated antigen was found by reacting serafrom two patients with giant cell tumor of bone with cellsderived from their tumors, using autologous serum asintermediate reactant and fluorescein-conjugated goat antihuman IgG as final reactant. Approximately 40% of theplump, spindle-shaped cells that formed the backgroundstroma of these tumors possessed the antigen; however, itwas not present on giant cells. Fluorescence was muchgreater than that on similarly stained cells from 4 osteogenicsarcomas, suggesting that the antigenic density on cellsfrom giant cell tumor was greater than that on cells fromosteogenic sarcoma. Antibodies in sera from giant celltumor patients and osteogenic sarcoma patients showedspecific cross-reactivity. Stromal cells of giant cell tumorswere established in culture and retained tumor-associatedantigen, whereas giant cells failed to divide and detachedfrom the flask within two weeks. Intensity of fluorescence(antigenic density) decreased with progressive passage 1evels, but a larger percentage of cells showed fluorescence. Atthe tenth passage, all cells bore tumor-associated antigen.Cultured cells that were injected s.c. into mice formedprogressively growing nodules, the cells of which weremorphologically indistinguishable from stromal cells of theoriginal tumor; all cells retained tumor-associated antigen,but antigenic density had decreased to about one-seventh ofthe value found originally. No giant cells were present in thenodules.

INTRODUCTION

What parameters dictate whether a given tumor willmetastasize? The answer to this question is not fullyunderstood at this time, but certainly 1 factor, host immuneresponse, plays an important role in this phenomenon. Host

1 This work was supported in part by USPHS Grant HD-0S894, by

Contract E 73-2001-NOI-CP-3-3237 with the Virus Cancer Program of theNational Cancer Institute, and by American Cancer Society GrantIM-I6

2 Recipient of a NIH Postdoctoral Fellowship (5 FO 2 AI-53320). To

whom reprint requests should be addressed, in care of the Department ofDermatology, University of California San Francisco, San Francisco,Calif.94143.

3 Recipient of Faculty Research Award PRA-88 from the American

Cancer Society.Received November 4, 1974; accepted June 6, 1975.

immune response could be modulated by 2 factors, immunocompetence and tumor antigenicity. We tested thelatter hypothesis by studying 2 closely related osseustumors. Osteogenic sarcoma is I of the most malignantosseous tumors known; death usually occurs from pulmonary metastases. In contrast, giant cell tumor of bone isoften considered clinically benign, in that pulmonary metastases (and death) are unusual. Although the primary lesionin both tumors is equally destructive, the 5-year survivalrate after surgical removal of the primary lesion is greaterthan 95% in giant cell tumor (4), as compared to 5 to 20% inosteogenic sarcoma (17). These 2 tumors are clinically andhistologically related, in that they affect a similar age range,occur in the same locations (21), and the backgroundstromal cells appear very similar morphologically (9). Giantcell tumor, inadequately radiated, can recur as osteogenicsarcoma (4).

The 2 tumors can be differentiated microscopically by therelative numbers of giant cells, which are large, multinucleated (25 to 100 nuclei) cells, and by the relative amounts oftumor osteoid; all osteogenic sarcomas have significantamounts of osteoid, and 35% of giant cell tumors have someosteoid (4). In view of the altered prognosis provided by thepresence of a significant percentage of giant cells, theirorigin and function are of great interest. It is not known ifthe giant cells in giant cell tumor represent a component ofthe neoplasm or whether they are part of a host response totumor. It is also not known whether the stromal cells, whichare actually the major cellular component of the tumor, arenormal or malignant.

The studies reported here were designed to determinewhether any of the cellular components of this tumorpossessed a tumor-associated antigen and, if so, which cellscarried the antigen. Additional studies on these cells weredesigned to determine whether cells bearing tumorassociated antigen could be established in culture and couldproduce tumors in immunosuppressed mice. The tumorassociated antigen on these cells was compared by quantitative indirect immunofluorescence with that on cells fromosteogenic sarcoma as to cross-reactivity and antigendensity.

MATERIALS AND METHODS

Preparation of Tissues. All specimens were obtained atsurgery immediately after biopsy or curettage or weredissected from the affected limb within 15 mm after

2520 CANCER RESEARCH VOL. 35

Quantitative Immunofluorescence Studies of the Tumor Antigenbearing Cell in Giant Cell Tumor of Bone and Osteogenic Sarcoma'

Vera S. Byers,2 Alan S. Levin,3 James 0. Johnston, and Adeline J. Hackett

Departments of Medicine [V. S. B.] and Dermatology [V. S. B., A. S. L.] University of California San Francisco, San Francisco 94143: WesternLaboratories, Oakland 94609 [A . S. L.]; Department ofOrthopedics, Kaiser Foundation Hospital, Oakland 94611 [J. 0. J.]; and School ofPublic Health,University of California Berkeley, Berkeley, California 94720 [A. I. H.]

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Quantitative Immunofluorescence in Giant Cell Tumor

amputation. Normal tissue was obtained by biopsy of skinor muscle. Under sterile conditions, specimens were placedin culture medium (Eagle's minimal essential medium withantibiotics) and transported to the laboratory at 4°.

Single-cell suspensions of tumors were prepared bycarefully trimming away all grossly appearing nontumortissue from the outside of the tumor section and rubbing thespecimen through a 250-gauge stainless wire mesh (Industrial Wire Products, Los Angeles, Calif.). Alternatively,sections from tumor or from normal tissue (skin or muscle)were incubated at 37°overnight in Dulbecco's modifiedmedium containing 10% fetal calf serum and collagenase, 1mg/ml (Worthington Biochemical Corp. Freehold, N. J.).Control experiments showed that both methods of obtainingsingle-cell suspensions from giant cell tumors yielded equivalent results. All single-cell suspensions were washed 3 timesin HBSS4 after preparation and were distributed onto slideswith a cytocentrifuge. Similar portions from the same tissuewere embedded in OCT tissue-embedding fluid for frozensections, (Miles Laboratories, Elkhart, Ind.), frozen, cutinto 4-@zmsections, and placed on slides (Clay Adams GoldSeal No. 3050). Coverslip monolayers ofcultured cells wereprepared as described below. All slides for fluorescencestudies were dried for I hr at room temperature, fixed in95% ethanol:methanol (9: 1), and air dried. Unstained slideswere stored at —70°until use.

Staining of Tissues. For this study, the indirect immunofluorescence technique was used. Duplicate samples ofappropriate single-cell suspensions, tumor sections, andnontumor tissue sections were prepared for immunofluorescence as follows. The samples were overlaid with theintermediate reactant, incubated for 30 mm at roomtemperature in a moist chamber, washed 3 times for 10 mmin PBS, pH 7.2, and damp dried. They were then overlaidwith the final reactant, a 1:20 dilution of fluoresceinisothiocyanate-conjugated goat antiserum to human a, ‘y,K,A, or@ chains, produced and rendered monospecific byappropriate absorption in our laboratory ( 11), again incubated for 30 mm, washed, and damp dried. The specimenswere mounted in phosphate-buffered glycerol, and coverslips were placed before examination by fluorescence microscopy.

“Controllevel―or “background―fluorescence was determined by using PBS as the intermediate reactant. Controlsincluded samples with an intermediate reactant of normalhuman serum diluted 1:2 in PBS or unlabeled goat antihuman IgG diluted 1:20 in PBS. “Experimental level―fluorescence was obtained from slides treated with anintermediate reactant of appropriate serum obtained frompatients at dilutions of 1:2 through 1:20 in PBS. Additionalslides of each tissue were prepared with an intermediatereactant of serum absorbed with autologous tumor-derivedcells or with autologous normal skin or muscle cells. These

‘Theabbreviations used are: HBSS, Hanks' balanced salt solution; IFI,index of fluorescence intensity [this should not be confused with Fl,fluorescence index, used to express the percentage of positive versusnegative cells in preparations stained by the indirect method (5)]; PBS,phosphate-buffered saline (0.01 M phosphate buffer in 0.9% NaCI solution, pH 7.2).

sera were absorbed by incubating equal volumes of packedcells and aliquots of serum for I hr at 37° and thenovernight at 4°. Serum was separated from cells bycentrifugation and used at the same dilutions as were thenonabsorbed sera. The final reactant for both sets of slides,control and experimental levels, was I of the fluorescein-labeled goat antisera described above. Results are at the 1:2dilution of test serum unless otherwise stated. Every experiment included autologous normal tissue as a specificitycontrol.

Membrane Immunofluorescence. Similar staining procedures were carried out to detect the presence of tumorassociated antigens on membranes of viable single-cellsuspensions from giant cell tumors. A single-cell suspensionfrom freshly removed tumor was prepared by rubbing tissuethrough a stainless steel screen and allowing debris to settleout. The cells were washed twice, adjusted to 3 x 10°/mI,incubated in suspension at 4°for 30 mm with a 1:10 dilutionoftest serum, washed twice in cold HBSS by centrifugation,and incubated for 30 mm at 4° with a 1:20 dilution offluorescein-labeled goat anti-human IgG in PBS. Blockingcontrol experiments were done on these viable single-cellsuspensions by incubating the suspensions at 4°with a I :10dilution of nonfluorescent goat anti-human IgG for 30 mmjust before incubation with fluorescein-labeled antiserum.

Determination of Intensity of Fluorescence.Control andexperimental fluorescence were measured with a LeitzOrthoplan incident lighting fluorescence microscope fittedwith a 100-watt high-pressure xenon light source and aMPV I microphotometer (E. Leitz, Inc., New York, N. Y.).A cell was centered in the viewing field, and the MPVdiaphragm was stepped down until only a single cell wasilluminated. The cell was then subjected to an excitationenergy source provided by the 100-watt xenon bulb filteredthrough BG 38, BG 12, and KP 490 filters. Light wasreflected from the 490/5 10 dichroic mirror so that theexcitation beam peaked at around 490 nm. Emitted lightwas then filtered through the dichroic mirror and through a510-nm suppression slide filter. All readings were made atx400. The intensity of the emitted beam was read on adigital picoammeter. The machine was calibrated such thatthe “darkcurrent― (the reading obtained with all diaphragms closed) was 0.5 x 10@ (±0.05 x l0@) amp. Atthis setting, with diaphragms in the reading position, themicroscope slide alone produced a reading of about 0.7 x10° amp.

First, a section of the specimen stained with hematoxylinand eosin was viewed to identify the cellular components ofthe tissue and their organization. Next, a similarly stainedsingle-cell suspension was viewed to determine the relativenumbers of the various cells in the preparation. Finally, asample of the same single-cell suspension stained withfluorescein-labeled conjugates was viewed by phase microscopy and transmitted visible light.

The cell to be measured was selected under phasemicroscopy and centered in the viewing field. The transmitted light was discontinued, and the cell was epi-illuminatedwith the excitation beam; the intensity of light emission at awavelength of 510 nm was then determined by allowing thephotometer to range and stabilize and by obtaining a

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V. S. Byers et a!.

measurement within 10 sec. Continuous illumination causedbleaching to become apparent after 30 sec, as evidenced bya gradual drop in values. Duplicate slides were preparedand used as necessary.

Measurements were taken of at least 3 representativecells on each slide, and each experimental and controlpreparation was prepared and measured in duplicate. Themean value is reported.

IFI. Our results are expressed as the IFI, a numberobtained by dividing the intensity of fluorescence in amp ofindividual cells that showed fluorescence in the experimentaltest by that of the same type of cells obtained from thecontrol preparation. An IFI of I equates with no differencebetween cells on experimental and control slides; preparations with an IFI below 3 were considered negative on ascale of 0 to 4+ by conventional immunofluorescence.

Tissue Culture. Samples from biopsy specimens of tumorand normal tissue obtained as described above were mincedinto 2-mm cubes and incubated at 37°overnight in Dulbecco's modified medium containing 10% fetal calf serum andcollagenase, 1 mg/mi. The cultures were changed to collagenase-free medium the next day, and medium was changedtwice weekly. When confluent, cultures were divided byadding 10 ml of a trypsin:EDTA solution (Grand IslandBiological Co., Grand Island, N. Y.) at 37°.Five mm later,cells were washed and seeded into 3 new flasks. At Passages5 and 10, cells were also seeded into Leighton tubescontaining coverslips for immunofluorescence studies. Tumor-derived cells were isolated by the Owens differentialtrypsinization technique (14), which successively decreasedthe number of fibroblasts (and increased the proportion oftumor antigen-bearing cells) at each passage. At Passage 5,cultures were frozen in dimethyl sulfoxide and stored inliquid nitrogen. Cells at Passage 5 were used for allexperiments reported here unless otherwise indicated.

Immunosuppressed Mice. Random-bred I 2-week-oldSwiss NAMRU mice (Naval Biomedical Research Laboratory, Oakland, Calif.) were given i.p. injections of0.25 ml ofantithymocyte serum (Microbiological Associates, Bethesda, Md.) on 3 consecutive days during the 1st week andtwice weekly during the next 2 weeks. After the 2ndinjection, 1.8 x 106 cultured tumor-derived cells from the5th passage were injected s.c. into the intrascapular regionof the neck. Nodules were excised 2.5 weeks after inoculation. The morphological appearance of cells was examined,and frozen sections of the nodules were prepared forexamination by immunofluorescence as described above.

RESULTS

Histopathological Appearance of Specimens

Slides prepared from single-cell suspensions and sections of giant cell tumors from 2 patients and osteogenicsarcomas from 4 patients were stained with hematoxylinand eosin (Fig. 1). The tumors of Patients I and 2 were classified independently as Grade III giant cell tumors by 3orthopedic pathologists. Sections of each giant cell tumorrevealed very aggressive background stroma consisting of

plump, spindle-shaped cells with pleomorphic nuclear patterns and a lack of polarity. There were moderate numbersof giant cells intercalated in this background, and tumorosteoid was present in both. The stromal cells appeared tobe of 2 types. Approximately 60% were benign-lookingmonocytic cells without nuclear mitotic figures: the remaining 40% were smaller, hyperchromatic cells with highmitotic activity. The same cell populations were easilydistinguished on stained slides made from single-cell suspensions of the tumors. The multinucleated giant cells were thelargest cells in the preparation; they were roughly 80 Mm indiameter. The stromal cells were of 2 sizes, either 9 to 12 Mmor 12 to 14 Mm, with the smaller cells having hyperchromatic nuclei. This distinction was not readily apparentunder phase microscopy. Tumor osteoid was not obvious insuch preparations. The giant cells and stromal cells weremore nearly spherical than they appeared on sections, butthey were easily identified.

The 4 osteogenic sarcomas were composed of anaplasticcells that were producing tumor osteoid in bizarre neoplasticpatterns (Fig. I). Some were also producing chondroidsubstrate tissue. The anaplastic cells ranged in shape fromround to spindle shaped. All were growing in disorganizednonpolar patterns. One osteogenic sarcoma (Patient B) hada few lymphocytes in situ. Stained preparations of singlecell suspensions from these tumors revealed a homogenouspopulation of cells very similar in size and morphology tothe background stromal cells in the giant cell sarcoma. Thefew lymphocytes were easily differentiated from these cellsby size (they were 6 to 8 Mm in diameter) and morphology.

Standardization of Fluorescence Technique

Comparison of Single-Cell Suspensions and TissueSections. Fixed, single-cell suspensions of tumor-derivedcells were the most satisfactory preparations for measurement of intensity of fluorescence because the fluorescence ofan isolated cell could be measured. By comparison, sectionsof tumor tissue were technically inferior because isolatedcells could not be centered uniformly in the diaphragm. Thepattern of fluorescence in cells from single-cell suspensionsresembled the pattern usually termed. “cytoplasmic―fluorescence. The entire cytoplasmic area of the cell showedfluorescence, and the fluorescence was evenly distributedover the cell except for the darker area over the nucleus.Occasionally, nucleolar or nuclear fluorescence was detected on culturedcellsexaminedby this technique,butnone of the tissues or sera reported here demonstrated suchpatterns.

Membrane Fluorescence. Because the entire cell wasflattened and fixed on slides made from single-cell suspensions, any tumor-associated antigens detected by test seracould have either been cytoplasmic or membrane associated. At least part of the fluorescence was membraneassociated, because test serum from a patient with osteogenic sarcoma and IgG x-type antitumor antibodies (previously shown to react against osteogenic sarcoma and giantcell tumor) produced membrane-type fluorescence whenreacted at 4°against viable cell preparations from 2 giant

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Quantitative Immunofluorescence in Giant Cell Tumor

cell tumors. Membrane immunofluorescence could not bequantitated because the bleaching effect occurred toorapidly. The cells that showed fluorescence in these preparations appeared to be the same as the plump, spindle-shapedstromal cells that also showed fluorescence in the fixedpreparations from single-cell suspensions (Fig. 2). Blockingcontrol experiments were done, B-lymphocytes and giantcells showed no or very weak fluorescence, whereas membrane immunofluorescence was seen in about one-half of theplump, spindle-shaped cells that were morphologically(under phase microscopy) the same as those forming thebackground stroma of the tumor.

Dilution of Serum. Experiments using giant cell tumorsand osteogenic sarcomas as substrates and various dilutionsof test serum as the intermediate reactant (Chart 1) demonstrated that fluorescence intensity on tumor-derived cellswas directly proportional to antibody concentration up tothe level of antibody saturation. Accordingly, at least 2 andusually 3 dilutions of each test serum were used asintermediate reactants with each tumor to ensure that thevalue reported represented the fluorescence intensity atsaturating levels of antibody. The final reactant, fluorescein-conjugated goat anti-human IgG, produced maximumfluorescence at a dilution of 1:20 and was used routinely atthat concentration.

Control Studies. The results of a typical experimentalprocedure are shown in Table I. Each experimental seriesalso included preparations of normal tissue from the samedonor that were stained and measured following the sameprocedures used for tumor-derived tissue. Preparations ofnormal tissue produced IFI between 0.95 and 1.6 with bothautologous and homologous sera.

Preparations ofeach tumor stained with PBS as intermediate reactant (control level fluorescence) were carefullyscanned for the presence of B-lymphocytes that mightproduce fluorescence on the experimental preparations.Although there were a few lymphocytes in 1 of the tumorsreported here, they were easily differentiated from the largerplump, spindle-shaped cells characteristic of stromal cells inthe tumor. Absorption of test serum with tumor tissue (butnot absorption with normal tissue) reduced fluorescenceintensity of cells in all experimental preparations so treatedby more than 50%, indicating that the measured cells werereacting with test serum and not directly with the fluorescein-conjugated anti-IgG. Additional controls included useof normal human serum or of unlabeled goat anti-humanIgG as intermediate reactant. The values of these preparations did not differ from the values of the control levelfluorescence preparations, in that variations among the 3types of preparations were no greater than those amongduplicate slides of the same preparation. Unlabeled goatanti-human IgG used as intermediate reactant also insuredthat autologous antibody was not fixed to tumor-derivedcells before preparation.

Tumor-associated fluorescence was considered significantif (a) the intensity of fluorescence of cells in the experimental preparation was greater than 3 times that of cells in thecontrol preparation, and (b) the tumor-absorbed serumsignificantly (at least 50%) reduced fluorescence intensity of

f0

‘I,

‘U

‘U

A.

4

SERUM DILUTION

Chart I . Intensity of fluorescence of 3 cells on each slide of a single-cellsuspension of osteogenic sarcoma, stained with various dilutions ofautologous serum as the intermediate reactant and with saturatingconcentrations (1:20 dilution) of fluorescein-conjugated goat antiserum tohuman IgG as the final reactant (see “Materials and Methods― for details).

these cells. In most cases, absorbed serum reduced fluorescence of such cells by 90%. In this study sera were onlyabsorbed once. Variations in fluorescence intensity amongindividual cells of the same type in the same control orexperimental preparation were no greater than 5% (Table1). Variations among a patient's cells of the same type ondifferent slides were less than 10%.

Results of Experimental Studies

FluorescenceStudies. Cells from both giant cell tumorswere prepared for immunofluorescence as described above,using various dilutions of autologous serum as the testserum in the experimental preparations. Giant cells fromboth tumors had very small increases in fluorescencebetween control and experimental fluorescence preparations, but approximately 40% of the plump, spindle-shapedcells that formed the background stroma of each tumorshowed bright fluorescence in the experimental preparations(Fig. 2). It could not be conclusively determined whether thecells showing fluorescence and, therefore, bearing tumorassociated antigen were the same as the hyperchromaticcells identified on the preparations stained with hematoxylinand eosin, but their distribution and relative proportionswere very similar. Attempts to stain cells after immunofluorescence studies were unsuccessful because detailed cellmorphology was disrupted.

The antibodies directed against these tumor-derived cellswere tumor specific, because they were removed by absorption with tumor tissue but not by absorption with normaltissue (skin or muscle) from the same donor. They wereclassified as IgG-K type because the fluorescence was seenonly when fluorescein-labeled antibody to human IgG orfluorescein-labeled antibody to immunoglobulin-bound Kchains was used as the final reactant.

The mean IFI of giant cells was less than 3.0 in all cases

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IntermediatereactantFinal reactantMeasurement°l0'x

ampSlide

IMeanSlide 2MeanMeanof

2 slidesIFlNoneNoneAutofluorescence0.29

0.280.300.290.24

0.240.260.250.27PBSFluorescein-conjugated

anti-human IgG (1:20)Controllevel fluorescence0.43

0.400.380.400.44

0.410.400.410.41Normal

humanserum (1:4)Nonspecific

binding offluorescein-conjugatedanti-lgG0.49

0.420.440.450.53

0.550.490.520.48Unlabeled

goat antihuman lgG (1:4)Autologous

antibody fixedto tissue before staining0.38 0.36

0.390.370.430.42

0.410.420.40Test

serum ( I :4)°Antitumor antibodies3. 182.962.822.993.42

3.233.403.353.177.7Test

serum (1:8)Effect ofdilution2.272.042.812.372.35

2.042.142.182.275.5Test

serum, tumorabsorbed (1:4)Specificity

of antitumorantibody0.78 0.80

0.730.77ND'1.8Test

serum, tumorabsorbed(1:8)Effect

ofdilution0.830.820.780.81ND2.0

V. S. Byers et a!.

Table I

Results of representative studiesperformed on single-cell suspensionsof tumor-derived cellsfrom a patient with osteogenicsarcoma using autologousserum

a All measurements were taken with coverslips in place. At the settings used for these studies, the slide and coverslip alone produced a value of about 0.7

x 10@ amp.P1 Each test serum was used at dilutions of 1:2, 1:4, and 1:8. Dilutions of 1:2 and 1:4 gave the same values. Serum absorbed with normal tissue from the

same donor yielded equivalent values.C ND, not done.

(Table 2). The IFI of the smaller stromal cells was 3 1.5 inone patient and 17.4 in the other. The IFI of the largerstromal cells was less than 3. There was no gradation offluorescence intensity within each population of cells in eachtumor; cells showed either bright fluorescence or very littleor no fluorescence. Cells from osteogenic sarcomas exammed by this technique had much lower IFI; all were below 7.There was also no gradation of fluorescence intensityamong the cells in these osteogenic sarcomas; all cells in theexperimental preparations showed the same level of fluorescence.

Comparison of IFI of Osteogenic Sarcoma and Giant CellTumor. Sera from 2 osteogenic sarcoma patients and fromgiant cell tumor Patient 1 were reacted against single-cellsuspensions of cells from the 4 osteogenic sarcomas and the2 giant cell tumors described above. Dilutions of thepatient's serum were used as intermediate reactant, andfluorescein-conjugated anti-IgG was the final reactant(Table 3). Controls included normal tissue from eachpatient as well as cells from I prostate carcinoma, 1

hypernephroma, I mammary carcinoma metastatic to bone,1 Ewing's sarcoma, 1 chondrosarcoma, and I reticulum cellsarcoma. Serum from giant cell tumor Patient I producedtumor-specific fluorescence of homologous tumor-derivedcells and of cells of all 4 osteogenic sarcomas tested. Serafrom 2 osteogenic sarcoma patients reacted with the smallerstromal cells in the giant cell tumors as well as with the cellsfrom osteogenic sarcoma. Measurements with each serumallowed the ranking of tumors as to antigenic density; theranking was almost the same with each serum. Only IFI isshown in Table 3, but identical rankings were obtained whenabsolute values (in amp) were used, because all control levelfluorescence values were quite similar. No serum produceda fluorescence index greater than 2 on cells from normaltissue or from the 3 control carcinomas or 3 controlsarcom as.

Giant Cell Tumor in Culture. In culture, cells from bothgiant cell tumors showed primary migration resulting in adense monolayer of plump, spindle-shaped cells and hundreds of multinucleated giant cells. Ten to 14 days after

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MethodGiant

cellsPlump, spindle-shapedstromalcellsFluorescenceFluorescence(1O@

x amp)IFI(l0' xamp)IFISingle

cell MeanSingle cellMeanPatient

IBackground(control) fluorescence8.02

7.267.20 7.507.32

7.546.487.11Test

serum'20.618.219.8 19.52.6208

22424122431.5Test

serum, tumor-absorbed13.112.113.9 13.01.79.71

9.5510.39.851.4Patient

2Backgroundfluorescence9.3 1

9.739.87 9.649.42

8.768.688.95Test

serum24.022.022.8 22.92.4164

14416015617.4Test

serum, tumor-absorbed19.318.018.8 18.71.923.5

24.025.4 24.32.7

Fluorescence index with serumfromOsteogenicOsteogenicGiant

cellsarcoma,sarcoma,tumor,Source

ofcellsPatient CPatient DPatientI@Osteogenic

sarcoma, PatientANDbND3.8Osteogenicsarcoma, Patient B4. 16.15.2Osteogenicsarcoma, Patient C4.03.17.0Osteogenicsarcoma, Patient D4.86.311.6Giant

cell tumor, PatientI8.310.931.5Giantcell tumor, Patient23.517.2NDMuscle,

PatientANDND0.9Skin,PatientBNDND1.0Muscle,PatientC1.11.01.2Skin,PatientD1.11.01.1

Quantitative Immunofluorescence in Giant Cell Tumor

Table 2

Quantitative immunofluorescence measurements ofsingle-cell suspensions of2 giant cell tumors

a The intermediate reactant was a 1 :4 dilution of autologous serum (tumor absorbed for certain experiments), and the final

reactant was an antibody excess of a 1:20 dilution of fluorescein-conjugated antiserum to human lgG.

Table 3

Quantitative immunofluorescenceofsingle-cell suspensionsof tumor-derivedand normal cellsPatient's serum was the intermediate reactant; in this series of experiments, the sera were not

absorbed. The final reactant was a I :20 dilution of fluorescein-conjugated antiserum to human IgG.

a Test serum from giant cell tumor Patient 2 was also reached against 2 of the osteogenic

sarcomas with similar results.@ ND, not done.

initiation ofculture, the giant cells began detaching from theflask, rounding up, and floating to the surface of themedium. The plump, spindle-shaped cells from both tumorscontinued to divide and grow. Coverslip preparations of celllines derived from the tumor (at Passages 5 and 10) andfrom the skin of Patient I (at Passages 4 and 5) werestained as described above using autologous serum. Cellsin cultures derived from tumor showed fluorescence,whereas those in cultures derived from skin did not(Table 4). At Passage 5, the majority of the cultured cells

derived from tumor showed fluorescence; at Passage10, all did. The fluorescence intensity, however, droppedfrom 31.5 in primary tumor-derived preparations to 9.2in cultures of tumor-derived cells at Passage 10.

Growth of Tumor Cells in Mice. Cultured tumor-derivedcells (Passage 5) of Patient I were injected into mice thathad been immunosuppressed by injections of antithymocyteserum. One week later a nodule was palpable at the site ofinoculation in 3 of 5 mice. The nodules were excised 1.5weeks later, at which time they were about 2 mm in

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Source ofcellsControl

fluorescence(l0'x amp)

Singlecell MeanTest

fluorescenceSingle

cellSerumusedas (l0@'x

intermediate reactant― amp)Mean(IO'x

amp)IFICultured

tumor cells(I0thpassage)0.53 0.53

0.58 0.54Patient

I 4.075.39

5.585.019.2Osteogenic

sarcoma 4.75Patient D 4.633.794.398.1Patient

D absorbed 3.52with D's skin cells 3.714.293.847.2Patient

D absorbed I .53with D's tumor cells I .38

1.961.623.0Cultured

skin fibroblasts(5th passage)0.44 0.39

0.41 0.41Patient

I 1.231. I2

1.111.152.8Patient

D 0.870.94

0.820.882.1Patient

D absorbed 1.47with D's skin cells 1.50

0.931.303.1a

All preparations were overlaid with a 1:20 dilution offluorescein-conjugated antiserum to humanIgG.

V. S. Byers et a!.

Table 4

Quantitative immunofluorescence of cultured skin cells and tumor-derived cells from Patient I

diameter. Sections were stained with hematoxylin and eosin;other sections were prepared for indirect fluorescence usingserum from Patient 1 (autologous serum). The stainedpreparations showed nests of cells embedded in adiposetissue. These cells appeared similar to the plump, spindleshaped cells in the original tumor and to those seen inreticulum cell sarcomas (Fig. 3). There was no evidence ofdevelopment of giant cells in these human tumor-derivedcells grown in mice, and there was no formation of osteoid.The intensity of fluorescence (IFI, 4.8) was not as strong asthat of cells from the original tumor, but all cells showedfluorescence.

DISCUSSION

Giant cell tumors of bone are made up of multinucleatedgiant cells intercalated on a background stroma of plump,spindle-shaped reticular cells and smaller pleomorphichyperchromatic cells. The giant cells are morphologicallyand histochemically similar to those present in chronicinflammatory reactions such as foreign body reactions andchronic infections (20). The larger stromal cells havenormal, well-organized nuclei and closely resemble histi

ocytes. These make up between 40 and 60% of thebackground stroma. The nuclei of these cells are quitesimilar to those in giant cells, and some investigators havepostulated that they fuse to form tumor-derived giant cells(8, 19). Several studies substantiate the hypothesis thathistiocytes (macrophages) can fuse in vitrO and in vivo toform giant cells (20). The 3rd cell type also forms part of thestroma but is smaller and has a hyperchromatic nucleus anda high mitotic index. Other investigators using electronmicrographic techniques have found the same 3 cellpopulations in giant cell tumor of bone (H. Paschall,personal communication).

We found IgG-K-type antibodies directed against cellsderived from giant cell tumors in the sera of 2 patients withgiant cell tumor. These antibodies reacted with about 40%of the stromal cells in the 2 tumors but not with the rest ofthe stromal cells or with giant cells. We therefore suggestthat the stromal cells expressing tumor-associated antigensare malignant cells, whereas the cells that do not expresstumor-associated antigens are normal histiocytes, some ofwhich fuse to form giant cells.

We conducted additional studies to determine which celltype met other criteria for a malignant cell. In culture, thegiant cells had no mitotic activity; 14 days after culture, all

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Q uantitative Immunofluorescence in Giant Ce!! Tumor

had detached from the flask, leaving only stromal cells.Indirect immunofluorescence demonstrated that, by the10th passage, all cells remaining fixed to the flask retainedthe tumor-associated antigen; this antigen was not presentin cultures of skin-derived cells, and antibodies against itcould be removed by absorption with osteogenic sarcomaderived cells.

Wheil cells from these cultures were injected into immunosuppressed mice, palpable nodules appeared 1 week laterat the site of injection; these nodules continued to grow untilexcised. Growth in immunosuppressed mice is 1 criterionfor judging malignancy of injected cells (I). The cellsforming nodules in our experiments appeared morphologically similar to the smaller hyperchromatic stromal cells ofthe original tumor; all of these cells bore tumor-associatedantigen, and no giant cells were seen. Nodules are sometimes seen after cultured cells derived from normal humantissues are injected into mice treated with antithymocyteserum. These reactive nodules, however, are obviouslydifferent in that they appear 1 or 2 days after inoculationand disappear within the week (1).

Gallardo el a!. (3) also found that giant cells detachedfrom the flask shortly after culture, whereas stromal cellssurvived. Their cells were grown on a nutrient agar platformat the 5th passage; some multinucleated cells formed, which,they suggested, were syncytial aggregates of stromal cells,implying that this mechanism of formation in vitro wasduplicated in vivo (3). We found no giant cells in the nodulesgrown in mice.

Although giant cell tumor of bone and osteogenic sarcoma cross-reacted, the fluorescence intensity of the cellsderived from giant cell tumor was far greater than that ofcells from osteogenic sarcoma, regardless of whether theserum detecting the antigen was from a patient withosteogenic sarcoma or a patient with giant cell tumor. Thisobservation has been made by others (12, 18). The LeitzMPV I microphotometer offered a method to quantitatethis difference. It has been shown by other workers inimmunofluorescence that the fluorescence intensity ofa cellis directly proportional to the concentration ofantigen in oron that cell. The substrates used in such studies have rangedfrom red cells coated with 1gM (10) to virus-infected cells(6). The fluorescenceintensityis alsodirectlyproportionalto the concentration of antibody when the substrate used iskept constant (7, 13).

Although we could not manipulate the concentration ofantigen in the tumor, we did verify that, in this system, thefluorescence intensity of a preparation was directly proportional to the concentration of the test serum used asintermediate reactant. Using saturating concentrations(antibody excess) of 3 test sera (from 1 patient with giantcell tumor and 2 with osteogenic sarcoma), we compared theantigenic density of 2 giant cell tumors with that of 4osteogenic sarcomas. The IFI was calculated for eachtumor-serum combination. The relative rankings of these 6tumors were the same, regardless of which serum was used(Table 3). This demonstrates that each antiserum crossreacted with the others. The extent of cross-reactivity is

uncertain because cross-absorptions were not performed inthese studies. Serum from Patient C gave the lowest set ofmeasurements and serum from Patient 1 gave the highest,suggesting that more than 1 set of antigens may be sharedand that serum from Patient C had antibodies against fewercommon antigens than did serum from Patient I. In ourstudies the tumors were ranked by IFI; however, theabsolute fluorescence intensity in amp of the experimentalpreparations gave the same results. The advantage of IFI isthat tissues with widely dissimilar control levels can becompared. In addition, IFI allows comparison betweentumor-serum combinations prepared and examined at different times. The antisera studied were tumor-specificbecause cells from autologous normal tissue had low lFIand because activity of antiserum was removed by absorption with tumor tissue but not normal tissue. The antiseramay be specific to osteogenic sarcoma and giant cell tumorbecause they did not react with cells derived from severalcarcinomas or from Ewing's sarcoma, chondrosarcoma, orreticulum cell sarcoma. However, too few other sarcomaswere studied to establish this point firmly.

In a previous report, we demonstrated cross-reactivitybetween osteogenic sarcoma and giant cell tumor on thecellular level (2). In this report we have shown cross-reactivity on a humoral level as well. However, there was adifference between the tumor antigen-bearing cell in giantcell tumor and that in osteogenic sarcoma; cross-reactiveantigens were much more dense on the cells in giant celltumor. This greater antigenic density could account for themore favorable prognosis associated with giant cell tumor,because the antigenic density may stimulate a vigorous hostresponse that confines disease to a single location andprevents metastatic events. The giant cells may be amanifestation of this response. Similar findings have beenreported by other investigators in animals. Prehn (15, 16)hypothesized that the effect of immunity on tumor cells isbiphasic, in that a minimal immune response to tumor cellsaccelerates growth of tumor, whereas a strong response iscytotoxic. Yutoku et a!. (22) recently demonstrated thatsmall doses of tumor cells injected i.v. into allogeneic miceresulted in lethal tumor growth in 90% of the animals,whereas doses 10-fold larger produced tumor rejection and70% survival. Cell-mediated immunity was much weaker inthe mice receiving low doses of tumor cells than in thosereceiving high doses; they suggested that the low dosecaused “immunostimulation― and the high dose producedimmune rejection.

These studies have similarities to the clinical situationreported here, except that we compared relative antigenicdensity on a cell-by-cell basis rather than absolute numbersof cells, and we found that cells with high antigenic densitiesoccurred in tumors associated with 90% survival of patients,whereas those with low antigenic densities were associatedwith 10% survival. We suggest, on the basis of the datapresented here, that at least some giant cell tumors of bonerepresent a variant of the same pathological process thatoccurs in osteogenic sarcoma and that the very differentclinical course is a result of host immune response.

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V. S. Byers et a!.

10. Killander, D., Levin, A., Inoue, M., and Klein, E. Quantification ofImmunofluorescence on Individual Erythrocytes Coated with VaryingAmounts of Antigen. Immunology, 19: 151-156, 1970.

11. Levin, A. S., Fudenberg, H. H., Hopper, J. E., Wilson, S. K., andNisonoff, A. Immunofluorescent Evidence for Cellular Control ofSynthesis of Variable Regions of Light and Heavy Chains ofImmunoglobulins G and M by the Same Gene. Proc. NatI. Acad. Sci.

U. S.,68: 169-171,1971.

12. Mukherji, B., and Hirshaut, Y. Evidence for Fetal Antigen in HumanSarcoma. Science, 181: 440-442, 1973.

13. Nairn, R. C., Herzog, F., Ward, H. A., and DeBoer, W. G. R. M.Microphotometry in Immunofluorescence. Clin. Exptl. Immunol., 4:697-705,1969.

14. Owens, R. B. Glandular Epithelial Cells from Mice: A Method forSelective Cultivation. J. Nail. Cancer Inst., 52: 1375- 1378, 1974.

15. Prehn, R. T. The Immune Reaction as a Stimulator ofTumor Growth.Science, /76: 170-171, 1972.

16. Prehn, R. T. Perspectives on Oncogenesis: Does Immunity Stimulateor Inhibit Neoplasia? RES J. Reticuloendothelial Soc., 10: 1- 16, 1971.

17. Price, C. H. G., and Jeffree, G. M. Metastatic Spread of Osteosarcoma. Brit. J. Cancer, 28: 515-524, 1973.

18. Priori, E. S., Wilbur, J. R., and Dmochowski, L. ImmunofluorescenceTests on Sera of Patients with Osteogenic Sarcoma. J. NatI. CancerInst.,46: 1299-1308,1971.

19. Schajowicz, F. Giant-Cell Tumors of Bone (Osteoclastoma): APathological and Histochemical Study. J. Bone Joint Surg., 43A:1-29, 1961.

20. Spector, W. G. The Macrophage: Its Origins and Role in Pathology.Pathobiol. Ann., 4@33_f4 1974.

21. Troup, J. B., Dahlin, D. C., and Coventry, M. B. The Significance ofGiant Cells in Osteogenic Sarcoma: Do They Indicate a RelationshipBetween Osteogenic Sarcoma and Giant Cell Tumor of Bone? Proc.Staff Meetings Mayo Clinic, 35: 179- 186, 1960.

22. Yutoku, M., Fuji, H., Grossberg, A. L., and Pressman, D. AnExperimental Model for Evaluation of Factors in Tumor Escape fromImmunological Attack. Cancer Res., 35: 734-739, 1975.

CANCER RESEARCH VOL. 352528

ACKNOWLEDGMENTS

We thank Merrill Knapp and Eddie Bautista for expert technicalassistance and Dr. Helene Smith and Dr. John Riggs for excellent criticismand suggestions. We are indebted to Dr. H. Hugh Fudenberg for financialsupport.

REFERENCES

1. Arnstein, P., Taylor, D. 0. N., Nelson-Rees, W. A., Huebner, R. J.,and Lennette,E. H. Propagationof Human Tumors in AntithymocyteSerum-Treated Mice. J. Nail Cancer Inst., 52: 71-84, 1974.

2. Byers, V. S., Levin, A. S., Hackett, A. J., and Fudenberg, H. H.Tumor-Specific Cell-Mediated Immunity in Household Contacts ofCancer Patients. J. Clin. Invest., 55: 500—513, 1975.

3. Gallardo, H., de Lustig, E. S., and Schajowicz, F. Growth andMaintenance of Human Giant-Cell Bone Tumors (Osteoclastomas) inContinuous Culture. Oncology, 24: 146-159, 1970.

4. Goldenberg, R. R., Campbell, C. J., and Bonfiglio, M. Giant-CellTumor of Bone: An Analysis of Two Hundred and Eighteen Cases. J.Bone Joint Surg., 52A: 619-664, 1970.

5. Gunvên, P., and Klein, G. Membrane Immunofluorescence. In: H.Busch (ed), Methods in Cancer Research, Vol. 8, pp. 177-200. NewYork: Academic Press, Inc., 1973.

6. Horan, M., Horan, P. K., and Williams, C. A. Quantitative Measurement of SV-40 T-Antigen Production. Exptl. Cell Res., 91: 247-252,1975.

7. Irvine, W. J., Chan, M. M., and Williamson, W. G. The Application ofMicrophotometry to the Indirect Immunofluorescent Antibody Technique Using the Human Gastric Parietal Cell Antigen-AntibodySystem. Clin. Exptl. Immunol., 4: 607-617, 1969.

8. Jaffe, H. L., Lichtenstein, L., and Portis, R. B. Giant Cell Tumor of

Bone: Its Pathologic Appearance, Grading, Supposed Variants andTreatment. Arch. Pathol., 30: 993-1031, 1940.

9. Johnston, J. (ed), The Pathology of Bone Tumors and Metabolic BoneDiseases, p. 129. Oakland, Calif.: Kaiser Foundation Hospital, 1972.

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Q uantitative Immunofluorescence in Giant Cell Tumor

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Fig. 1. A, a 4-@zmsection of giant cell tumorof bone from Patient I . Several giant cells areintercalated over a background stroma of hyperchromatic and nonhyperchromatic (arrows)plump, spindle-shaped cells. H & E, x 250. B,another area of the same section of giant celltumor, showing an area of tumor osteoid, x 250;inset, same area, x 540. This patient and the 2nd

patient with giant cell tumor have remainedtumor-free more than 2 years after amputationwith no further therapy. C, a 4-sm section ofprimary osteogenic sarcoma of Patient B, showing tumor osteoid, x 250. This patient hadpulmonary metastases at the time of surgery. Twoof the other patients with osteogenic sarcomadeveloped metastases after surgery; the 4th patient is receiving chemotherapy.

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Fig. 2. A , single-cell suspension of giant cell tumor of bone from Patient 1. Solid arrows, hyperchromatic cells; hollow arrow, nonhyperchromaticcells, both in background stroma. H & E, x 250. B, indirect immunofluorescence of a single-cell suspension of giant cell tumor, using patient's serum asintermediate reactant and fluorescein-labeled antiserum to human IgG as final reactant. Nonfluorescent giant cells in background are not visible; stromalcells showing fluorescence are found both in clusters (as shown here) or, more usually, as single cells. x 540. C, lower magnification of same preparation.Photomicrograph is overexposed so that nonfluorescent giant cells are visible, as well as stromal cells showing fluorescence (arrow). x 250. D, membraneimmunofluorescence of viable antigen-bearing cells from giant cell tumor of bone (see “Materials and Methods― for details).

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Fig. 3. Murine nodule resulting frominoculation with cultured cells derived fromtumor of Patient 1. A , H & E stain of a 4-smsection. x 250. B, indirect immunofluorescence of specimen from nodule, using Patient l's serum as intermediate reactant andfluorescein-labeled antiserum to human IgG

as final reactant. x 250. C, higher magnification ofA. x 540.

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1975;35:2520-2531. Cancer Res   Vera S. Byers, Alan S. Levin, James O. Johnston, et al.   Osteogenic SarcomaAntigenbearing Cell in Giant Cell Tumor of Bone and Quantitative Immunofluorescence Studies of the Tumor

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