Journal of Immunological Methods, 7 (1975) 347--358 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

ASSAY OF CELL-MEDIATED CYTOTOXICITY BY TRITIATED THYMIDINE LABELED TUMOR CELLS

ROGER J. FERGUSON, JAMES H. McMASTER and CARL R. WEINERT, Jr. Department of Orthopaedic Surgery, 986 Scaife Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, U.S.A. (Received 27 August 1974, accepted 31 December 1974)

A microtechnique for quantitating cell-mediated cytotoxicity using tritiated thymidine labeled target cells is presented. Target cell incorporation of tritiated thymidine is augmented by using methotrexate to arrest endogenous thymidine synthesis. Complete solubilization of labeled cells is accomplished by oxidizing samples to tritiated water and carbon dioxide prior to quantitation in a scintillation counter. This versatile technique, using various combinations of lymphocytes, sera and target cells, permits the simultaneous comparison of 500 or more individual samples.

INTRODUCTION This s t u d y was initiated to d e v e l o p a m e t h o d o f r a p i d l y q u a n t i t a t i n g a large n u m b e r o f s a m p l e s in a c e l l - m e d i a t e d c y t o t o x i c i t y assay. Since R o s e n a u a n d M o o n ( 1 9 6 1 ) first devised an in vitro assay for c e l l - m e d i a t e d c y t o t o x i c i t y m a n y t e c h n i c a l r e f i n e m e n t s h a v e b e e n m a d e in an e f f o r t to f u r t h e r quantit a t e this p h e n o m e n o n . T e m p o r a l f l u c t u a t i o n s in c y t o t o x i c activity o f a p a t i e n t ' s l y m p h o c y t e s as well as v a r i a t i o n s in t h e g r o w t h characteristics o f each t a r g e t cell s u b c u l t u r e d e m a n d an assay p r o c e d u r e w h i c h p e r m i t s the s i m u l t a n e o u s c o m p a r i s o n o f a large n u m b e r o f t e s t s a m p l e s a n d a p p r o p r i a t e c o n t r o l s . T h e n u m b e r o f samples w h i c h can be t e s t e d in a n y c e l l - m e d i a t e d c y t o t o x i c i t y e x p e r i m e n t is l i m i t e d b y : (1) t h e n u m b e r o f l y m p h o c y t e s t h a t can be o b t a i n e d f r o m a p a t i e n t w i t h o u t c o m p r o m i s i n g his well-being; (2) t h e t i m e a n d e f f o r t inv o l v e d in q u a n t i t a t i n g each s a m p l e . A logical m e a n s o f s i m p l i f y i n g and q u a n t i t a t i n g t h e c y t o t o x i c i t y r e a c t i o n is r a d i o i s o t o p i c labeling o f t a r g e t cells. T h e release o f r a d i o a c t i v i t y i n t o t h e c u l t u r e m e d i u m or r e t e n t i o n o f label b y surviving t a r g e t cells can t h e n be quantitated. R a d i o i s o t o p e s u s e d t o label t a r g e t cells have i n c l u d e d c h r o m i u m s 1 (51 Cr), i o d i n e 1 2 s (1 2 si) and t r i t i u m (3H); h o w e v e r , e a c h o f t h e s e i s o t o p e s have certain u n d e s i r a b l e features, and to d a t e n o n e has gained w i d e s p r e a d p o p u larity. Ideally, to be suitable f o r use as a t a r g e t cell label, a r a d i o i s o t o p e

348 should be permanently bound to the target cell by a known metabolic pathway and released at cell death without reutilization by the surviving cells. Also, it should be incorporated in quantities which permit clear differentiation of labeled samples from background radiation, expose target cells to minimal radiation damage and present no radiation hazard to laboratory personnel. The technique developed in this study uses a low concentration of methotrexate (MTX) in the culture media to suppress endogenous thymidine synthesis, thus increasing target cell incorporation of exogenous [ 3H] thymidine (3HT). The MTX block in conjunction with the extremely high recovery efficiency of the 3HT label following oxidation permits clear differentiation of labeled target cells from background radiation. This method permits the simultaneous testing of 500 or more individual samples. A detailed description of this labeling technique with an example of its application in the study of cell-mediated c y t o t o x i c i t y in human osteosarcoma is presented. MATERIALS AND METHODS

Preparation of target cells Tumor and autologous skin specimens were obtained at surgery and placed in Gey's solution containing 1% penicillin and streptomycin. Representative sections of the t u m o r were prepared for tissue culture as described by Green (1971). Cell monolayers were detached from culture flasks by treatment with 0.25% trypsin for 10--15 min at 37°C. The cell suspension was centrifuged, the cells washed with Gey's solution, resuspended in 1--2 ml of Gey's solution, and a suspension containing 1000 cells/50 pl ,was prepared. One thousand cells were dispensed into each well of a Falcon 3040 microtest plate using a Hamilton PB-600-1 repeating dispenser. To each well was added 0.1 ml of basal media (Eagle's) with Earle's salts (EBME) containing 20% fetal calf serum (FCS) and the cells were incubated for 72 hr at 37°C in a humidified 5% CO2--air atmosphere. EBME media containing MTX (3 × 10 .4 mM), and 3HT (10 pCi/ml) was prepared, 0.1 ml of this solution was added to each well, and incubation continued for an additional 24 hr. The radioactive media was then removed by gentle suction and the wells rinsed three times with Gey's solution and air dried (fig. 1).

Determination of lyrnphocytes--target cell ratio The number of labeled cells in each well must be known so that a constant l y m p h o c y t e to target cell ratio can be maintained throughout each experiment. The number of counts per minute (cpm) measured in the scintillation counter for each labeled cell was determined for fibroblasts (F5-73) and

349

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osteosarcoma cells (T1-73) in the following manner. Three thousand cells were inoculated into tissue culture flasks (25 sq cm) and incubated for 72 hr in EBME with 20% FCS in a 5% CO2--air environment. The media was then r e m o v e d and 4 ml of media containing MTX and 3HT in the concentrations described above was added to each flask. Incubation was continued for 24 hr after which time the media was removed and the cells washed to remove a d h e r e n t radioisotope. Ten combusto-cones {Packard I n s t r u m e n t Company) each containing 1, 2, 3, 4, 5, 6, 8, or 10 t housand cells were oxidized and c o u n t e d . The average cpm for 10 samples was then divided by the n u m b e r of cells per co n e to obtain the c o u n t per minute per cell (cpmpc). When target cells were labeled for a c y t o t o x i c i t y assay, the average cpm f o r 10 wells was d e t e r m i n e d for each target cell line prior to adding lymphocytes. This p e r m i t t e d qua nt i t a t i on of the l y m p h o c y t e / t a r g e t cell ratio.

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Fig. 2. P l o t o f C P M vs cell n u m b e r t o d e t e r m i n e t h e m e a n c o u n t p e r m i n u t e p e r cell ( c p m p c ) f o r a f i b r o b l a s t cell l i n e F 5 - 7 3 a n d a n o s t e o s a r c o m a cell l i n e T 1 - 7 3 . T h e n u m b e r s in p a r e n t h e s i s are t h e m e a n c p m p c f o r 10 s a m p l e s .

Cy to toxicity assay A double matrix was used for each c y t o t o x i c i t y assay with l y m p h o c y t e c y t o t o x i c i t y on the horizontal axis compared to serum effect on the vertical axis. To each well containing labeled target cells 0.1 ml of a 1 : 7 dilution o f heat inactivated test serum was added. After incubation for 1~ hr at 37°C the serum was removed and peripheral blood l y m p h o c y t e s , separated by Hypaque--Ficoll density gradient centrifugation (Boyum, 1968), and 0.1 ml o f EBME with 20% FCS were added to each well. The lymphoc y t e - t a r g e t cell ratio was 50 : 1. After 45 min of incubation an additional 0.1 ml o f EBME with FCS was added and the plates incubated for 60 hr. After incubation each well was washed three times with Gey's solution to remove l y m p h o c y t e s and detached target cells, and air-dried. Aerosol spray (Aeroplast) was applied to each microtiter plate to prevent mechanical det a c h m e n t o f the remaining target cells. The lower third of each well was removed, placed in a combusto-cone, and oxidized in a Packard 306 Oxidizer. The oxidation pr oduct , tritiated water (3H2 O), in 10 cc of Monophase 40 scintillation fluid (Packard) was c o u n t e d in a scintillation counter.

Calculation o f cytotoxic index The l y m p h o c y t e c y t o t o x i c index (CIL) was calculated using the formula:

CIL =

(CPM-Lo) -- (CPM-Lx) (CPM-Lo) × 100%

351

where: CPM-Lo was t h e c p m in wells w i t h o u t l y m p h o c y t e s and CPM-Lx was t h e c p m in wells t o w h i c h t e s t l y m p h o c y t e s were a d d e d . A similar c a l c u l a t i o n was p e r f o r m e d t o e v a l u a t e t h e e f f e c t s o f sera u p o n t a r g e t cell 3 H T i n c o r p o r a t i o n using t h e f o r m u l a : (CPM-So) (CPM-Sx) (CPM-So) X 100% -

CIs =

-

where: CPM-So was t h e c p m in wells w i t h o u t sera a n d CPM-Sx was the c p m in wells t o w h i c h t e s t sera was a d d e d . RESULTS

The effects o f methotrexate P r e l i m i n a r y studies i n d i c a t e d t h a t 1 0 0 0 cells per well a n d 72 hr o f incubat i o n were o p t i m u m f o r growing h u m a n o s t e o s a r c o m a cells in m i c r o t i t e r wells. T h e e f f e c t o f various c o n c e n t r a t i o n s o f M T X u p o n t h y m i d i n e i n c o r p o r a t i o n b y h u m a n o s t e o s a r c o m a cells a n d skin f i b r o b l a s t s was t h e n determ i n e d (table 1). O s t e o s a r c o m a cells g r o w n in t h e p r e s e n c e o f 1.6 × 10 -4 m M M T X i n c o r p o r a t e d 1 3 5 0 + 1 1 8 c p m as c o m p a r e d t o cells g r o w n w i t h o u t M T X w h i c h i n c o r p o r a t e d 514 + 21 c p m . F i b r o b l a s t s g r o w n in the s a m e c o n c e n t r a t i o n o f M T X h a d an activity level o f 1 5 0 6 -+ 115 c p m , while f i b r o b l a s t s g r o w n w i t h o u t M T X i n c o r p o r a t e d 4 3 0 + 75 c p m . This repres e n t e d an a c t i v i t y level a p p r o x . 35 t i m e s t h e b a c k g r o u n d level o f 40 c p m f o r b o t h o s t e o s a r c o m a cells and f i b r o b l a s t s . I n c r e a s i n g t h e M T X c o n c e n t r a t i o n b e y o n d 1.6 × 10 -4 m M did n o t significantly alter the u p t a k e o f 3HT.

TABLE 1 The u p t a k e of [s H] t h y m i d i n e by osteosarcoma cells and fibroblasts during three days of i n c u b a t i o n was significantly increased in the presence of m e t h o t r e x a t e . Concentrations above 3.2 × 10 -4 mM did n o t lead to additional uptake of [3H] thymidine. METHOTREXATE CONCENTRATION

DAYS OF INCUBATION

OSTEOSARGOMA CELLS

FIBROBLASTS

514 -+ 5 ~

4 3 0 -+18

1,6 x 10-4mM

1350 ± 29

1506 + 28

3,2 x 10"4rnM

1480 ± 38

1105 -+ 52

6,4 x 10"4raM 1,6 x 10-3raM

1386 -+ 39

1537 --4"39

1545 -+ 28

1620

0,0

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Assay of cell-mediated cytotoxicity by tritiated thymidine labeled tumor cells.

A microtechnique for quantitating cell-mediated cytotoxicity using tritiated thymidine labeled target cells is presented. Target cell incorporation of...
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