[CANCER RESEARCH 32, 1042-1044, May 1972)

Preservation of Cellular Antigenicity of Tumor Cells by theUse of Formalin Fixation

Walter P. Drake, Peter C. Ungaro, and Michael R. Mardiney, Jr.

Section of Immunology and Cell Biology, NIH, National Cancer Institute, Baltimore Cancer Research Center, Baltimore, Maryland 21211

SUMMARY

Immunization of rabbits with formalin-fixed EL-4lymphoma cells of the C57BL/6 mouse was found to yieldantitumor sera equivalent in cytotoxicity to that obtainedwith fresh, viable cells. In addition, indirectimmunofluorescence studies indicate that antigenicdeterminants on the cell membrane remain intact followingfixation. Formalin fixation of tumor cells may be a usefulmethod in immunotherapy, as well as an aid to the elucidationof antigens associated with the tumor cell membrane.

INTRODUCTION

The production of high-liter antisera to intact cells hasnecessitated repeated administration of antigen over a periodof several weeks (8, 10). When particular cells are readilyavailable, this necessity has not resulted in significantdifficulty. However, when cells must be obtained by extensiveoperations, the ability to preserve cells harvested at the time ofa single procedure becomes desirable. Preservation of cells withmaintenance of surface antigens is further required for theproduction of appropriate antisera.Viable cells have been an important prerequisite for the

production of adequate antilymphocyte serum (3, 12).Although antiserum has been harvested after immunizationwith frozen cells preserved in a fashion incompatible with themaintenance of viability (4), it has not been demonstrated thatantigenic determinants are preserved, and the effectiveness ofsuch an antiserum is questionable.Preservation of antigenic sites by the formalin fixation of

erythrocytes has been demonstrated (5, 6). These studies showthat this method of preservation does not alter the binding ofantibody to the cell membrane. Additional studies havedemonstrated that formalin-fixed, antigen-sensitized redblood cells result in greater reproducibility in thehemagglutination reaction than that obtained by the use offresh cells (2).This report is concerned with the use of formalin fixation in

the preservation of antigenic determinants of mouse tumorcells. Preservation of tumor cells in this way permits theproduction of high-liter antisera from cells oblained at a singlepoint in lime and provides a slore of cells of prolongedstability for use in related experiments.

Received January 25,1972;accepted February 14, 1972.

MATERIALS AND METHODS

Cell Preparations. Carcinogen-induced EL-4 tumor cells (7)in the ascitic form were harvesled from Ihe peritoneal cavilyof C57 black mice by repeated lavages with 0.85% sterile NaClsolution (NIH Media Unit, Belhesda, Md.).For frozen cell preparalions, cells were quick-frozen in an

acetone-Dry Ice bath and subsequenlly lhawed in a waler balhat 37°.

Formalinized cell preparations were made by washing thecells with 0.85% NaCl solulion and cenlrifuged at 1000 rpm(Mislral Model 6L centrifuge) for 15 min. Twenty-five volumesof 10% buffered formalin were added lo Ihe packed cells,which were Ihen vorlexed and left at room temperature for 12hr. Cells were then washed twice with 0.85% NaCl solulionand slored al 10°until ready for use.

Reagents. For 10% buffered formalin, the followingcomponenls were combined lo a final volume of l liier, pH7.2: 100 ml of 40% formaldehyde, 4 g NaH2PO4-H2O, and6.5 g Na2HPO4. Trypan blue solulion, 0.2%, was usedIhroughoul.Immunizations. New Zealand female rabbils weighing aboul

12 Ib (Animal Resources, Woodsboro, Md.) were immunizedwilh 109 cells (eilher spleen lymphocytes or EL-4 tumor cells)on Days 0, 14, and 21, and antisera were harvested on Day 28by cardiac puncture. After inaclivalion of complemenl byhealing al 56° for 45 min, anlisera were assayed for

cyloloxicily against C57BL/6 spleen and EL4 tumor cells.Assay for Cytotoxic Activity. The Irypan blue exclusion

lesi was ulilized lo ascerlain Ihe cyloloxic aclivily of thevarious anlisera (8). Spleen cells were prepared by pressing 3aseplically removed C57BL/6 spleens Ihrough a slainless sleelscreen in 5 ml of 0.85% NaCl solulion. After filtration througha filler (pore size, 150), Ihe cell suspension was subjecled lohypolonic lysis by addition of 20 ml of distilled water for 20sec, followed by the addition of 5 ml of Veronal-buffered0.85% NaCl solution, 5-fold concentraled (NIH Media Unii).This trealmenl was generally repealed after Ihe cell suspensionwas filtered and centrifuged at 1000 rpm (Mistral Model 6Lcentrifuge). The cell preparalions were finally resuspended inVeronal-buffered 0.85% NaCl solulion conlaining 15%heat-inaclivaled felal calf serum (NIH Media Unii) andwere > 95% viable.EL-4 lymphoma cells were oblained in ascitic form from

C57BL/6 mice and were > 95% viable. Bolh cell preparalionsconlained less lhan 15% red blood cells.All cell preparations were adjusted to 106/ml, and 0.1 ml of

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Formalin Fixation of Tumor Cells

cells was used for an assay. To this was added 0.1 ml ofappropriately diluted inactivated antiserum, and incubation at37°for 15 min with constant agitation followed. This mixturewas then cooled to 0°,after which 0.1 ml of fresh-frozen

guinea pig complement (Grand Island Biological Co., GrandIsland, N. Y.), diluted 2:1 with Veronal-buffered 0.85% NaClsolution: 15% fetal calf serum was added, and incubation wasallowed to continue at 37°for 45 min. The tubes were then

cooled and centrifuged at 1500 rpm (Mistral Model 6L),supernatant was decanted, and 3 drops of 0.2% trypan bluewere added. Tubes were vortexed and percentage of viablecells was determined by classification of 200 cells as stained orunstained.Cytotoxic antibody titer is expressed as the reciprocal of

that dilution of antibody that kills 50% of the cells in thecyto toxic assay.Immunofluorescence. Fluorescein-conjugated goat

anti-rabbit y-globulin and fluorescein-conjugated goatanti-human 7-globulin were obtained from BehringDiagnostics, Inc., Woodbury, N. Y. Antiserum, 0.1 ml, wascombined with 10 X IO6 cells and incubated for 20 min at37°.Preparations were then washed twice with 2 ml of 0.85%

NaCl solution by successive centrifugations at 1000 rpm for 15min. Fluorescein-labeled antiserum, 0.4 ml, was added, andcells were incubated as above. After being washed twice with0.85% NaCl solution, cells were finally resuspended in 0.4 mlof phosphate-buffered 0.85% NaCl solution-glycerin mountingmedium. A drop of suspension was placed on a glass slide, andthe coverslip was ringed with molten paraffin. Preparationswere examined with a Leitz Wetzler Orthomat optical system.After the microscope was focused with visible light, cells werescored for fluorescence under UV illumination.Positive fluorescence is defined by bright, distinct cells with

high intensity of fluorescence at the membrane. Negativefluorescence was observed when cells were not distinguishableunder UV illumination.

RESULTS

Trypan Blue Exclusion. Both fresh and formalinized cellsdemonstrate equal ability to exclude trypan blue dye (Table1). Cells subjected to freezing show a markedly reducedcapacity to withstand perfusion by the dye.Antisera Titers Obtained. Data in Table 2 indicate that, at

least with respect to cytotoxicity, both formalinized and freshcells provoke a similar immune response. Titers against EL-4cells of 1200 and 1750 obtained for fresh cells are comparableto titers of 1000 and 1500 obtained from animals immunizedwith formalin-fixed cells. In contrast, immunizations madewith frozen tumor cells led to cytotoxic titers of 400 and 500.Immunofluorescence. Table 3 presents the results of a

fluorescence assay used to detect antibody bound toformalinized cells. Antibody cytotoxic for EL-4 tumor cellsproduced from immunizations with either C57 spleen cells orEL-4 cells gave positive fluorescence. Neither of thefluorescein-labeled antibody stains bound to cells incubatedwith 0.85% NaCl solution only. The fact that EL4 cellsincubated with antisera to Group A red cells gave negative

fluorescence when stained with goat anti-human 7-globulin(fluorescein-labeled) further attests to the specificity of thisassay. Formalin-fixed cells gave somewhat more distinct andmore intense fluorescence than that obtained with fresh cells.Controls with 0.85% NaCl solution, however, were equivalent.

Table 1Trypan blue exclusion

Cells were harvested from the peritoneal cavity of C57BL/6 mice 7days after tumor transfer. After manipulations described in "Materialsand Methods," cells were stained with 0.2% trypan blue. Percentage ofintact cells was determined after counting of 200 cells and representsthat fraction that exludes the dye.

Cell preparation Intact cells (%)

EL4 cells, freshEL-4,cells, fixedEL-4 cells, frozen

<â!¢)H9810

Table 2Cytotoxic titers obtained from various cell preparations

Animals were immunized with 10' EL-4 cells, either fresh, frozen, orfixed. Titers are expressed as the reciprocal of that antiserum dilutionthat kills 50% of EL-4 cells in a cytotoxic assay. Repeat assays gaveidentical results ±10%.

Rabbit Cell preparation Titer against EL-4

123456EL-4 cells,freshEL-4cells,freshEL-4cells,frozenEL-4cells,frozenEL-4cells,fixedEL-4cells, fixed1250175040050010001500

Table 3Immunofluorescence

Cells were incubated with either 0.85% NaCl solution or antiserum,followed by staining with fluorescein-labeled antibody. Positivefluorescence was indicated by observation of distinct cells under UVillumination. For negative fluorescence, distinct cells were not observedafter the microscope was focused with visible light and aftersubsequent examination under UV illumination. Antisera used aredescribed more completely in "Materials and Methods."

Fixa-CellstionEL-4

+EL-4EL-4

+EL-4EL-4

+EL-4EL-4

+EL-4EL-4

+EL-4ABABFluores-

Antiserum GAR0 GAHcence0.85%,

NaCl solution+0.85%NaCl solution+0.85%NaCl solution+0.85%NaCl solution+Anti-C57BL/6

++Anti-C57BL/6++Anti-EL^++Anti-EL^t++Anti-A

+Anti-A+Anti-A

++0.85%NaCl solution +

°GAR, fluorescein-labeled goat anti-rabbit 7-globulin; GAH,fluorescein-labeled goat anti-human 7-globulin; +, positive fluorescence;-, negative fluorescence; AB, human red blood cells, type AB.

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W. P. Drake, P. C. Ungarn, and M. R. Mardiney, Jr.

DISCUSSION

Formalin-fixed cells maintain gross membrane structure tothe extent that they are capable of excluding try pan blue. Thetiters obtained after immunization of rabbits with these cellssuggest that their immunological potential is not compromisedand hence that antigenic determinants are largely intact. Theseformalinized cells are clearly not viable cells, yet they maintainimmunogenicity to a far greater extent than those cellsrendered nonviable by freezing and subsequent thawing.It is important to point out that, in many previous

experiments, immunization with fresh EL-4 cells has alwaysled to cytotoxic titers in the range of 1000 to 1750 against thetumor cells. Hence, it is significant that formalin-fixed cellsresult in antisera with equivalent cytotoxicity.The fluorescence data demonstrate that antigenic sites on

the EL-4 cell membrane are largely unchanged by fixation,since only antisera cytotoxic for these cells become bound.Both anti-EL-4 and anti-C57BL/6 spleen, in which there iscross-reactive cytotoxic activity against EL-4 cells, givepositive fluorescence after incubation with EL-4 tumor cellsand subsequent staining with fluorescent antibody (Table 3).The binding of antibody in this assay is specific, as shown bythe negative fluorescence obtained when EL-4 cells areincubated with anti-Group A red cell. As would be expected,nonviable cells have repeatedly been shown to take up thestain nonspecifically (1), and this was also observed in thissystem. The degree of fluorescence obtained with fixed cellswas somewhat more intense than that obtained with freshcells, although controls with 0.85% NaCl solution wereequivalent. This is probably due to the decrease in cellclumping following fixation, which would lead to betterexposure of the membranes to the various antisera.The above evidence suggests that formalin fixation of tumor

cells results in cell preservation without alteration ofmembrane antigenic sites. Although the use of liquid nitrogenfreezers for slow cooling, with concurrent use of preservativessuch as dimethyl sulfoxide (11), may result in immunizing cellpreparations of equivalent efficacy to that of fresh cells,formalin fixation is a simple technique requiring no specialequipment. In addition, with respect to animal tumors, a stockof cells can be harvested at a specific point in time,formalinized, and used as a constant source of immunizingcells with avoidance of possible immunogenic alteration of celllines maintained in vivo or in culture.This use of formalin to preserve cellular surface antigens

may also be of importance in permitting ready storage of

human tumor cells obtained at a single operation. These wouldthen be available for subsequent immunizations, which atpresent are an important part of specific immunotherapy (9).

ACKNOWLEDGMENTS

We thank Miss Eleanor Dukehart for her help in the preparation ofthis manuscript.

REFERENCES

1. Baldwin, R. W., Barker, C. R., Embleton, M. J., Glover, D., Moore,M., Pimm, M. V. Demonstration of Cell-Surface Antigens onChemically Induced Tumor. Ann. N. Y. Acad. Sci., 777: 268-278,1971.2. Daniel, T. M., Weyand, J.' G. M., Stavisky, A. B. Micromethods for

the Study of Proteins and Antibodies. IV. Factors Involved in thePreparation and the Use of Stable Preparations of Formalinized,Tannic Acid Treated, Protein-Sensitized Erythrocytes for theDetection of Antigen and Antibody. J. Immunol., 90: 741-750,1963.

3. Dempster, W. J. Freezing and Anti-Lymphocyte Globulin. Brit.Med. J.,4. 742, 1969.

4. Doak, P. B., Dalton, N. T., Meredith, J., Montgomerie, J. Z., andNorth, J. D. K. Use of Antilymphocyte Globulin after CadavericRenal Transplantation. Brit. Med. i.,4: 522-525, 1969.

5. Economidou, J. A Study of the Reactions between Certain HumanBlood Group Antigens and Their Respective Antibodies withSpecial Reference to the ABO System. Ph.D. Dissertation, LondonUniversity, London, 1966.

6. Gold, E. R., Lockyer, W. J., and Tovey, G. A. Use of LyophilizedFormal-Treated Red Cells in Blood Group Serology. Nature, 182:951-954,1958.

7. Gorer, P. A. Studies in the Antibody Response of Mice to TumorInoculation. Brit. J. Cancer, 4: 372, 1950.

8. Kim, C. A., and Reif, A. E. Immunization Schedules for PotentRabbit Antisera to Leukemia L1210. Cancer Res., 31: 7-11, 1971.

9. Mathé,G., Amiel, J. L., Schwarzenberg, L., Schneider, M., Cattan,A., Schlumberger, J. R., Hayat, M., de Vassal, F. ActiveImmunotherapy for Acute Lymphoblastic Leukemia. Lancet, 1:697-706, 1969.

10. Pichlmayr, R. Aspects in Production of Antilymphocyte Sera.Federation Proc., 29: 111-113, 1970.

11. Smith, M. J. A. Low Temperature Preservation of MouseLymphocytes with Dimethyl Sulfoxide. Blood, 23: 494-501,1964.

12. Wood, M. L., and Vriessendorp, H. M. A Comparative Study ofHeterologous Antimouse Lymphocyte and Antimouse ThymocyteSera Prepared by Two Different Immunization Methods.Transplantation, 7: 522-533, 1969.

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