- Email: [email protected]
Modification of the clonogenic assay for the detection of lymphokine activated killer cell activity
Journal of Immunological Methods, 128 (1990) 119-126
119
Elsevier JIM 05509
Modification of the clonogenic assay for the detection of lymphokine activated killer cell activity * Philip Scuderi ~, Linda Woo ~, Anita S.-F. Chong 2, Rosa Liu t and Sydney E. Salmon t From Arizona Cancer Center, University of Arizona College of Medicine. Tucson. AZ. U.S.A.. and 2 Rush Presbyterian St. Luke's Medical Center. Chicago. IL. U.S.A.
(Received 28 July 1989, revisedreceived30 November 1989. accepted 1 December 1989)
The soft agar clonogenic assay using [3H]thymidine uptake as an endpoint was adapted for the detection of h u m a n LAK activity against the Daudi lymphoma cell line and h u m a n tumor cells obtained by biopsy. Using Daudi cells as the target population the modified agar assay was more sensitive than the conventional 4 h SlCr release assay. Use of a single layer agar assay allowed the assessment of LAK cytotoxic/cytostatic activity against Daudi lymphoma cells after cell to cell contact, while a two layer syste,'n permitted evaluation of the role of soluble mediators in L A K / t a r g e t cell interactions, This study shows, that LAK cells can either kill or inhibit the proliferation of Daudi cells by two mechanisms: one which requires cell-to-cell contact, and a second via soluble mediators. As determined by the use of neutralizing antisera, the soluble factor(s) are not tumor necrosis factor and interferon-,/. Of the nine individual h u m a n tumor samples obtained by biopsy, 89% were sensitive to allogeneic LAK cells when the two populations were admixed. Of these nine tumors 44% were inhibited by LAK-derived soluble factors. The soft agar assay system should serve as a useful tool for determining the sensitivity of h u m a n rumors to L A K cells and for studying the mechanisms of LAK anti-tumor activity. Key words: Clonogenic assay; Lymphokin¢activated ,killer; Cytokine
Introduction The activation of h u m a n peripheral blood leukocytes (PBL) with interleukin-2 (IL-2) gives rise to a phenotypically heterogeneous population of lymphokine activated killer (LAK) cells
Correspondence to: P. Scud¢~'i,Arizona Cancer Center. University of Arizona. 1515 N. Campliell Avenue, Tucson, AZ 85724. U.S.A. * This research was supported by NCI Grants CA21839, CA17094, CA23074and Grant 8277-000000-1-0-YR-9301from the Arizona Disease Control Research Commission. Abbreviations: LAK, lymphokine activated killer; HTCA, human tumor clonogenic assay; TNF, tumor necrosis factor; IFN-y, interferon-y.
(Herberman et al., 1987). In vitro L A K cells are either cytotoxic or cytostatic to established tumor cell lines and to h u m a n tumor cells obtained by surgical excision. In short term isotope release :,ssays the killing of h u m a n tumor cells is not restricted by differences in major histocompatibility complex antigens displayed by the L A K and tumor cell populations and most tumor cells appear to be susceptible to cytolysis ( G r i m m et al., 1982). This non-restricted in vitro anti-tumor activity initially suggested that LAK cell therapy would be applicable to a broad spectrum of human malignancies. To date, however, L A K cell therapy has shown promise for only a fimited number of tumor types (Rosenberg et al., 1985, 1987). The discrepancy between the cytotoxic ac-
0022-1759/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
tivity of LAK cells in vitro and the efficacy of LAK cell therapy suggests that the interaction between effector and target cell populations as measured by the conventional 4 h 51Cr release assay may not be an accurate reflection of LAK cell anti-tumor activity. LAK cells have recently been reported to secrete cytotoxic factors distinct from either tumor necrosis factor or interferon-v (Hersh et al., 1989). The anti-tumor activity of these factors is only detectable in in vitro assay systems such as the soft agar clonogenic assay in which tumor ceils are exposed to soluble mediators for 48 h or more. Thus short term isotope release assays are an ineffective means of measuring anti-tumor activity dependent on LAK derived factors. The soft agar human tumor clonogenic assay (HTCA) is a technique which is widely used to test the activity and clinical utility of chemotherapeutic agents against human tumor ceils obtained by biopsy (Luedke et al., 1984). The HTCA has several distinct advantages over other methods of drug screening, it allows testing to be performed on a small number of tumor cells, the soft agar environment fosters the outgrowth of human t.~mor cells which may not proliferate in fiquid medium, it inhibits the growth of normal fibroblasts, and there is a good correlation between the sensitivity or resistance of tumor cells to a given agent in vitro and the utility or non-utility of that agent in vivo (Von Hoff, 1983). We have developed an approach for use of the soft agar assay for detecting LAK activity against human tumor cells. The system was initially developed using the LAK sensitive Daudi lymphoma cell line and was subsequently used to test the effect of LAK cells on human tumor cells ob= tained by biopsy. Our results suggest that in HTCA human tumor cells are susceptible to either LAK mediated cytolysis or cytostasis after cell to cell contact, in some instances by soluble factors secreted by the LAK cells.
Collection (Rockville, MD) and grown in complete tissue culture medium consisting of RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin and streptomycin. Human tumor cells used in this study which were obtained by biopsy for routine diagnostic purposes after informed consent and were either used immediately or viably frozen in liquid nitrogen until needed. PBL were collected from 24 healthy adult volunteers by venous puncture in heparinized tubes. Mononuclear leukocytes were obtained from heparinized whole blood after separation of Ficoll-Hypaque (Pharmacia, Piscataway, N J). LAK cells were produced by incubating PBL at a density of I x 106/ml in complete medium containing 1000 U / m l IL-2 at 37°C for from 4-7 days. In some experiments LAK cells were irradiated with from 10 to 50 Gray in order to test the effect of LAK proliferation on the assay system.
Reagents Recombinant IL-2 was a gift from Cetus (Emeryville, CA). Recombinant TNF and IFN-'~ were gifts from Genentech (S. San Francisco, CA). Rabbit antiserum specific for either human TNF or IFN-¥ was made by immunizing New Zealand White rabbits with the appropriate recombinant cytokine using established techniques (Scuderi et al., 1987). 51Cr release assay Daudi cells were labelled with 51Cr in the form of sodium chromate and the assays were carded out in round-bottomed 96-weU plates by adding 1 × 104 labelled Daudi cells to each well, followed by the appropriate number of LAK ceils. The plates were spun at 100 X g to facilitate effectortarget cell contact and incubated for four hours at 37°C in a humidified incubator. The percent specific release was calculated using the following formula:
Materials and methods
experimentalrelease-spontaneousrelease × 100 maximumrelease-spontaneousrelease
Cells and culture conditions The human B cell lymphoma cell line Dandi was obtained from the American Type Culture
The experimental release was obtained by incubating effector and target cells at various ratios. Maximum release was produced by adding 1 N
121 HC! to target cells and the spontaneous release was obtained from target cells incubated alone for 4 h at 37°C. All groups were assayed in quadruplicate. Human tumor cells Human tumors were surgically excised, minced into 10 mm 3 sections and placed in sterile hypoosmolar medium consisting of 69 ml of Dulbecco's balanced salt solution supplemented with 8% bovine serum albumin fraction V (Sigma Chemical Co., St. Louis, MO), 20 ml of a 5% solution of polyvinylpyrrolidone (Sigma) in deionized distilled water, 10 ml of 2% methylcellulose (Sigma) in distilled water and 1 ml of a 10% solution of tetrasodiumethylenediaminetetraacetic acid in distilled H20. For each ~ of tumor suspension, 0.5 ml of McCoy's medium containing 3 m g / m l porcine collagenase (Sigma) and 0.3 m g / m l DNase (Sigma) were added. The tumor tissue was subsequently disaggregated by gentle stirring at 37°C for 1 h. The tumor cell suspension was passed through sterile gauze and a 30/~m mesh screen to remove undigested fragments. The tumor cells were washed three times in McCoy's medium containing 10~ fetal calf serum. A single cell suspension was produced by gentle pipetting and ~he viability of each population was assessed by trypan blue due exclusion. Soft agar assay Tumor cells were suspended at a density of 1 × 10 s cells/ml in complete medium. 1 ml of tumor cell suspension was combined with an equal volume of complete medium containing the appropriate number of L A K cells. LAK cells which were stimulated with 1000 U / m l of IL-2 for from 4 - 7 days and were washed three times in Hanks' balanced salt solution (Irvine Scientific, Santa Ana, CA) before being combined with the tumor cells. After a 24 h incubation at 37°C in a humidified incubator 200 /~1 of a 3.5% solution of Sea-Prep 15/45 Agarose (FMC, Rockland, ME) was added. 200/~1 of this cell suspension was then added to each well of a round bottomed 96-well plate. The 0.35% agarose solution containing the cells was solidified by incubating the plate on ice for 30 mln. The assay plates were subsequently incubated at 37°C for 96 h and then pulsed with
3H-TdR by adding 10/~l of a 10/~Ci/ml solution to each well. The plates were then incubated for an additional 48 h. The cells were harvested by floating the assay plates for 2 rain in a 67°C water bath to melt the agarose followed by evacuation of the wells under vacuum and collection of the cells on cellulose acetate filter strips using a multiple automated sample harvester. To ensure that all the cells were collected the wells were repeatedly washed with warmed cae+/Mge+-free phosphatebuffered saline (PBS) supplemented with 37.4 r a g / ! porcine trypsin and 15 m g / l EDTA (Gibco, Grand Island, NY). The harvesting manifold was rinsed in PBS alone between each set of wells. The filter strips containing the immobilized cells were air dried and then immersed in Beckman ReadySole scintillation cocktail (Beckman, Fullerton, CA) and counted in a liquid scintillation counter. Controls included wells containing tumor cells alone and LAK cells alone. The percent inhibition was calculated using the formula: 1
(cffector + target)qam × 100 (effectorcpm) + (target clam)
In the two layer agar system the lower layer was created by solidifying 100 /~! of 0.35% agarose solution in complete rt~edium containing 5 × 104 tumor cells. The upper layer containing the appropriate number of LAK cells was subsequently added and solidified. Incubation times and 3HT d R labelling for the two layer system were the same as used in the single layer assay, except that the upper layer containing LAK cells was removed prior to the addition of isotope.
Results Comparison between L A K activity in the SICr release and soft agar assays For the purposes of comparing the sensitivity of the 51Cr and the single layer agar a~say, PBL were stimulated with IL-2 for 4, 5 and 7 days and then tested on Daudi cells at effector-to-target ratios of from 20 : 1 to 0.08 : 1. Fig. 1 shows that in both the conventional SlCr and the agar assay, 5 day LAK produced the maximum effect. Comparing the two assays, it is clear that the agar assay
122 TABLE I TRITIATED THYMIDINE INCORPORATION IN THE SOFT AGAR HUMAN TUMOR CLONOGENIC ASSAY (HTCA) a LAK : Daudi ratio
Experiment I LAK + Daudi
LAK alone
Experiment 2 LAK + Daudi
LAK alone
Experiment 3 LAK + Daudi
LAK alone
20 : 1
2,908.2 + 925.0
2,354.8 4-372.2
2,598.6 + 362.4
4,253.9 + 493.9
25,549.8 4-2,828.3
32,791.9 + 137.8
10 : 1
3,928.0 + 379.3
934.1 5:47.1
1,588.8 5:152.6
1,038.0 5:219..5
14,075.3 5:1,492.2
17,273.4 + 3,598.4
5 :1
9,809.0 5:4,246.0
463.8 + 66.2
1,101.8 + 212.4
621.4 + 153.4
8,692.3 4-893.5
5,947.9 5:145.9
2.5 : 1
3,030.5 4-1,267.0
190.4 5:69.4
548.5 + 103.7
319.2 5:101.1
4,020.4 + 348.9
1,793.0 5:589.3
1.25 : 1
8,929.0 + 6,159.3
109.4 + 48,2
1,300.6 + 255.4
212.4 + 52,7
9,270.5 :i: 2,181.3
549.3 + 83.1
0.63 : 1
29,603.2 5:1,641.4
85.7 + 39.0
21.905.8 5:2,015.3
101.0 + 21.8
53,281.7 5:2,571.2
383.0 5:327.5
0.31 : 1
36,983.0 + 4,124.2
97.3 + 62.9
58,076.8 + 7,549.9
82.3 + 39.4
133.072.5 + 7,985.0
602.5 :1:245.4
0.16:1
47,870.1 :t: 2,801.1
43.8 :i: 15.4
65,278.9 5:8,068.4
40.0 5:22.6
149,118.3 + 11,508.2
846.5 + 114.6
-
68,881.0 5:3,794.2
31.3 4-15.4
155,799.2 4-10,476.9
2,485.0 4-187.3
0.08 : 1 Daudi alone
47,161.5 5:3.104.6
68,243.4 4-4,091.2
154,415.3 4-2,980.5
a The values shown are the mean of four observations and are expressed as counts/min + standard deviation. LAK cells used in these experiments were produced by incubating human PBL in IL-2 for 5-days.
detects m o r e L A K activity at t h e h i g h e r effectorto-target ratios. T a b l e I s h o w s t h e effect o f a d d i n g t h r e e different L A K p o p u l a t i o n s to D a o d i cells. I n e a c h e x p e r i m e n t L A K cells i n d u c e d a c o n c e n t r a tion d e p e n d e n t decrease in t h y m i d i n e i n c o r p o r a tion. T o test t h e possibility that L A K cells were r e s p o n d i n g to t h e D a u d i cells b y i n c o r p o r a t i n g 3 H - T d R , s o m e L A K cell p r e p a r a t i o n s were irradia t e d with 1 0 - 5 0 G r a y w h i c h criminated t h y m i d i n e i n c o r p o r a t i o n . I n t h e 51Cr a s s a y irradiated L A K r e t a i n e d their cytolytic activity a n d in t h e a g a r a s s a y they p r o d u c e d a n inhibition o f 3 H - T d R c o m p a r a b l e to t h a t i n d u c e d 5 y u r . i ~ a d i a t e d L A K ( d a t a n o t shown).
Optimum length of LAK-Daudi coincubation I n the initial e x p e r i m e n t s 24 h c o i n c u b a t i o n o f L A K a n d D a u d i cells w a s arbitrarily c h o s e n a n d
t h e cells were p l a t e d in a g a r a l o n g with t h e c u l t u r e supernatants. To determine the optimum coincub a t i o n t i m e for L A K a n d D a u d i cells, t h e t w o p o p u l a t i o n s were either c o m b i n e d a n d i m m e d i a t e l y a d d e d to a g a r o r a d m i x e d a n d coincub a t e d for f r o m 3 h to 24 h a n d t h e n p l a t e d in agar. I n addition, t h e effect o f factors w h i c h were prod u c e d d u r i n g t h e c o i n c u b a t i o n period o n t h e D a u d i target cells w a s tested b y w a s h i n g s o m e o f t h e effectors a n d targets after c o i n c u b a t i o n a n d susp e n d i n g t h e m in fresh c o m p l e t e m e d i u m . Fig. 2 s h o w s t h a t t h e o p t i m u m ~ ? i b i t i o n w a s achieved a f t e r a 12 h c o i n c u b a t i o n . T h e s e e x p e r i m e n t s also d e m o n s t r a t e t h a t soluble factors p r o d u c e d d u r i n g t h e c o i n c u b a t i o n d o n o t play a role in t h e inhibition o f 3 H - T d R u p t a k e . I n t h e p r e v i o u s experiment~ t h e a d m i x t u r e of L A K a n d D a u d i at a ratio o f 2 0 : 1 followed i m m e d i a t e l y b y the a d d i t i o n o f t h e cells to a g a r
123 100
O--O 0--0
~-~_
A--A
4- DAY LAK 5 DAY LAK 7 DAY LAK
i1.
\
\\
I
0,01 z
oI 20:1
°'--"-o. \
\
•
\ \ . 10:1
.
. 5:1
.
.
2.S:1
"~0~8~:--~ 1,2S:1 0.63:1 0,S1:1 0.16:1 0.08:t
EFFECTOR : TARGET RATIO
Fig. 1. LAK cells produced by incubating 1 x 106 PBL/ml in complete RPMi medium containing 1000 U / m l recombinant
human IL-2 for the indicated length of time, were admixed with 1 xl04 5lCr-labelled Dandi target cells at the indicated ratios (upper panel). The lower panel shows a soft agar assay which measuresthe inhibitionof 3H-TdR uptake by LAK and Daudi cells at the indicated ratios. 1xl04 Daudi were admixed with LAK and incubated at 37°C for 24 h in complete medium. The cells were subsequently suspended in 0.35% agaros¢ in a 96-weU assay plate. Controls included 3H-TdR uptake by LAK alone at the various ratios used and by Daudi alone. One of two experimentsis shown and each value displayed represents the mean of four replicates+the standard deviation. Where standard deviation bars are not shown they are obscuredby the symbolsize.
were separated in a two layer system. In the two layer system Daudi cells were added to liquid agar which was then solidified on ice and then the LAK cells were over-layered in liquid agar. The upper liquid layer was subsequently solidified on ice and the assay plates were incubated under standard conditions. Under these conditions the two agar layers remain as distinct agar plugs throughout the course of the assay. After an initial 96 h incubation at 37°C the upper agar layer containing the LAK cells was removed under gentle vacuum and the remainir:$ lower layer was pulsed with 3H-TdR. The inhibition in thymidine uptake was equal when cells were either admixed in the same layer of agar or separated in the two layer system (data not shown).
Effect of cytokme-specific antisera on L A K activity Since the two layer HTCA studies s u ~ e s t e d the presence of a diffusible factor(s) which could inhibit thymidine incorporation, cytokine-specific neutralizing antisera were added to the agar to determine whether they could block the diffusible factor activity. Fig. 3 shows that the addition of 10,000 neutralizing umts of rabbit anti-TNF and anti-IFN-y either when the LAK and Daudi were initially admixed or following the twenty-four hour
10o ] E
ao~
20
resulted in a 31% inhibition in thymidine uptake. Under these conditions LAK and Daudi cells were suspended in the agar with few effector and target cells in physical contact with one another. The resultant inhibition suggested that soluble mediators may play some role in the reduced thymidine incorporation. To determine if soluble factors produced after the LAK and Daudi cells were added to a single layer of agar were responsible for the inhibition the two populations were either edmixed and immediately added to agar or they
ol
~ NO WASH P7~ WASH
1i
0 3 6 12 24 TIME EFFECTORS AhD TARGETS COINCUBATEDBEFORE PLATING (hr)
Fig. 2. LAK and Daadi cells were combined at a 20:1 ratio and either plated in agar immediately (0) or coincubated at 37°C for the indicated length of time before the addition of agar. After coincubation, LAK and Daudi were added to the agar either with the conditioned medium (NO WASH) or they were washed in Hanks" balanced salt solution and resuspension in complete medium before plating. One of two experiments is shown and each valut: represents the mean inhibition of 3HTdR uptake of four replicates 4- the standard deviation.
124 OVARIANCARCINOMA
coincubation did n o t effect the inhibition o f thymidine incorporation.
Sensitivity of human tumor cells obtained by biopsy to LAK activity in the single and double layer agar assays T h e u p p e r panel o f Fig. 4 shows that w h e n h u m a n t u m o r cells obtained b y biopsy were admixed with allogeneic L A K for 18 h a n d subsequently a d d e d to the single layer agar system, that eight o f the nine t u m o r cell populations were inhibited. O f the t u m o r types tested only a single l y m p h o m a was refractory to L A K activity in this assay. In contrast to the inhibition o f 3 H - T d R observed w h e n L A K a n d t u m o r were c o i n c u b a t e d a n d then a d d e d to agar, the separation o f effector a n d t u m o r cell p o p u l a t i o n s in two layers (lower panel) resulted in inhibition o f only four o f the nine populations. Interestingly the l y m p h o m a which was uneffected b y coincubation was susceptible to inhibition in the two layer system. E a c h t u m o r cell p o p u l a t i o n was tested with three different allogeneic L A K cell p o p u l a t i o n s a n d in each
Ill
pTA
0':1:t
10080-
~ 60
~,o 20 0
1i1i1i1i NRS
ANTI-TNF ANTI-IFN- T ANTI-TNF
+
ANTI-IFN-7
Fig. 3. LAK and Dandi cells were admixed at a ratio of 20:1 in complete medium alone and in medium containing either 20 /tl of normal rabbit serum or 10 ~tl each of the indicated cytokine-specific antisera. The ceils were incubated 24 h and then added to agar. LAK and Daudi which were initially coincubated alone received either cytokine-spefific antiserum or normal rabbit serum at the time of agar addition. Anti-TNF and anti-IFN-y used in these experiments each contained 1000 neutralizing units of activity/pl. One of four experiments is shown and the values displayed represent the mean of four replicates 4-the standard deviation.
LYMPHOMA E2D I-2-1 MELANOMA SARCOMA
401
00
LAK AND TUMORCELLSADMIXED10:1 RATIO
I I
40]
? [ ~ ADDEDWHEN LAK AND DAUDIWEREADMIXED E22~,t'DDEDWHEN LAK AND DAUDIWERE.ADDEDTO AOAR
l
_
LAK ANDTUMORCELLSSEPARATED10:1 RATIO Fig. 4. Ailogeneic L A K and I x l O 4 human tumor 'oclls obtained by biopsy were admixed at the indicated ratio, in-
cubated for 24 h at 37°C and added to agar (upper panel). The lower panel shows the effect of incubating LAK and human tumor cells separately for 24 h followed by plating the ceils in two separate agar layers. One of three experiments, each using the same tumor cell populations and different allogcneic LAK, is shown and the values displayed represent the mean of four replicate,-.4-the standard deviation.
o f the experiments similar t r e n d s were observed. Six o f the n i n e t u m o r cell p o p u l a t i o n s were tested in a 5tCr assay, however, the s p o n t a n e o u s isotope release was b e t w e e n 40 a n d 56% making the results o f these experiments u n i n t e r p r e t a b l e (data n o t shown).
Effec: of adding cytokine-specific antisera to the two layer agar system with a human lymphoma Sufficient n u m b e r s o f the l y m p h o m a , which was sensitive to b o t h the contact m e d i a t e d a n d soluble effects o f L A K cells, were available to test the role o f T N F a n d I F N - 7 in L A K mediated c y t o t o x i c / c y t o s t a t i c activity. Fig. 5 shows that a
125
6O
]
4o
[ [
20-
ANTI-TNF
I
ANTI--IFN--7
ANTI--TNF + ANTI-IFN-7
Fig. 5. Lymphoma cells were suspended in. complete medium containing 0.35~ agarose at a density of 1 ×105 cells/ml. The rabbit anti-TNF and anti-lFN-? were added at a final concentration of 10,000 U/well, 20 ;tl of normal rabbit serum was used as a control. One of two experiments is shown and the values displayed represent the mean of four observations+ the standard deviation.
suspension of L A K cells in agar containing 10,000 neutralizing units of anti-TNF and anti-IFN-7 either alone or in combination had no effect on the inhibition of 3H-TdR uptake by the lymphoma. In these experiments the upper layer containing immobilized LAK cells was removed at 96 h after which time the lymphoma cells in the lower layer were pulsed with 3H-TdR.
Discussion
This report demonstrates that the soft agar clonogeuic assay can be adapted as a sensitive and reproducible means of detecting L A K activity against human tumor cells. The agar assay has several advantages over the conventional 51Cr release assay. It is more sensitive than the isotope release assay at higher effector-to-target ratios. The enhanced sensitivity of the agar assay is probably due in part to the extended coincubation time within which LAK and tumor cells are in physical contact. Additional sensitivity may also occur because the agar assay detects cytotoxicity mediated by both cell contact and soluble factors. Thus the second advantage of the agar assay is that it can be used, in the two laver format, to distinguish between direct cell-mediated and secreted factor-
mediated cytotoxicity. LAK cell populations can secrete a variety of cytotoxic cytokines including T N F and IFN-y and the release of both T N F and IFN-y can be stimulated by human tumor cells (Chong et al., 1989a,b). L A K cells are also capable of secreting one or more unique cytotoxic cytokines distinct from T N F and IFN-'r (Hersh et al., 1989). The third advantage of the assay system is that the soft agar environment allows the screening of fresh human tumor biopsies which may preferentially grow when immob'flized in agar (Hamburger et al., 1978). Both the single layer and double layer agar systems described in this report should be adaptable for the study of other effector populations. In the present report heterogenous L A K cells were studied. The 3H-TdR uptake used as an endpoim in these assays was suitable for measuring tub, or cell proliferation because L A K deprived of IL-2 failed to proliferate and incorporate thymidine in our system. The introduction of IL-2 into the agar assay haduced LAK proliferation which was unpredictable and often exceeded that of tumor cell populations (data not shown). However, in related experiments we have determined that irradiation of LAK cells with up to 5000 rads can inhibit L A K proliferation but still permit LAK-induced inhibition of target cell 3H-TdR incorporation. Thus one limitation of the agar system is that the effector population must either not proliferate o~" they must take up thymidine at levels well below that of the tumor target cell population. This requirement may be met by macrophages and monocytes, but may not be possible when using T cells as the effector population. The experiments reposed here suggest that both the single and double layer agar system may be a useful tool for studying L A K cell mediated cytolysis. Our findings are consistent with pubEshed accounts of L A K activity measured in single layer agar assays using colony counting as an endpoint (Bradley et al., 1985; Klvin-Nelemans et al., 1989). Further, our system which uses 3H-TdR vptake as an end point may be adaptable for the study of cytotoxic and cytostatic activity produced by other cell types. The assays described here may also provide a means of studying both contact and factor mediated antitumor activity produced by a single effector cell population.
126 Referenee~ Bradley, E.C., Catino, J.J., Issell, B.F., Pniesz, B., Huslad, J.M., Dalton, T., Allegretta, M. and Mier, J. (1985) Cell-mediated inhibition of tumor colony formation in agarose by resting and interleukin 2-stimulated human iymphocytes. Cancer Res. 45,1464. Chong, A.S.-F., Aleksijevic, A., Scoderi, P., Hersh, E.M. and Grime.s, W.J. (1989a) Phenotypic and functional analysis of LAK cell clones: Ability of CD3 + LAK cell clones to produce IFN-gamma and tumor necrosis factor upon stimulation with tumor targets. Cancer Immunol. Immunotber. 29, 270. Chong, A.S.-F., Scuderi, I~., Grimes, W.J. and Hersh, E.M. (1989b) Tumor targets stimulate IL-2 activated killer cells to produce IFN-y and tumor necrosis factor. J. Immunol. 142. 2133. Grimm, E.A., Mazumder, A,, Zhong, A,Z. and Rosenberg, S.A. (1982) Lymphokine-activated killer cell phenomenon lysis of natural killer-resistant fresh solid tumor cells by interleuldn 2-activated autologoas human peripheral blood iymphocytes. J. Exp. Med. 155,1823. Hamburger, A., Salmon, S.E., Kim, M., Trent, J.M., Soehnlen, B.J., Aiberts, D.S. and Schmidt, H.J. (1978) Direct cloning of human ovarian carcinoma cells in agar. Cancer Res. 38, 3438. Herberman, R.B., Hiserodt, J., Vujanovic, N , Balch, C., Lotzova, E., Bolhvis, R., Golub, S., Lanier, L.L., Philfips, J.H., Riccardi, C., Ritz, J., Santoni, A., Sclunidt, R.E. and Uchidm. A. (1987) Lzmphokine-aetivated killer cell activity. Immunol. Today 8,178. Harsh, E.M., Scuderi, P., Grimes, W.J., Chong, .~ ' .F., Brailey, J.L., Gschwind, C.R. and Salmon, S.E. (l:~og) Cytotoxic and cytostatic activity of lymphokine activated killer
(LAK) cell supematants. Cancer lmmunol, lmmunother. 30, 65. KlvinoNelemans, H.C., Van der Harst, D , Van den Burgh, J.F., Van Luxemburg, S. and Brand, A. (1989) Comparison between clonogenic and cytotoxic assays for measuring LAK cell activity. Intern. J. Cell Cloning 7, 232. Luedke, D.W., Carey, F.J., Krishan, A., Chce, D., Chang, B., Franco, R., Niell, H.B., Jones, M.E., Kenady, D.E. and Zirvi, K. (1984) Interlaboratory reproducibifity of the human tumor colony forming assay (HTCA): The Southeastern Center Study G~oup (SECSG) experience. In: S.E. Salmon and J.M. Trent (Eds.), Human Tumor Cloning. Grune and Stratton, Orlando, FL, p. 1245. Rosenberg, S.A., Lotze, M.T., Muul, L.M., Leitman, S., Chang, A.E., Ettinghausen, S.E. Matory, Y.L., Skibber, J.M., Shiloni, J.M., Vetto, J.T., Seipp, C.A., Simpson, C. and Reichert, C.M. (1985) Observations on the systemic administration of autologous lymphnkine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. New Engi. £ Med. 313,1485. Rosenberg, S.A., Lotze, M.T., Muul, L.M., Chang, A.F., Auis, F.P., Leitman, S., Linehan, W.M,, Robertson, C.N., Lee, R.E., Rubin, J.T., Seipp, C.A,, Simpson, C.G. and White, D.E. (1987) A progress report on the treatment of 157 patients with advanced cancer using iymhokine-aetivated killer cells and interleukin-2 or high dose interleukin-2 alone. New Engl. J. Med. 316, 889. Scuderi, P., Sterling, K.E., Raitano, A.B., Grogan, T.M, and Rippe, R.A. (1987) Recombinant gamma interferon stimulates the production of tumor necrosis factor in vitro. J. Interferon Res. 7,155. Von Hoff, D.D., Clark, G.M., Stogdill, B.J., Sarnsdy, M.F., O'Brien, M.T., Casper, J.T., Mattox, D.E., Page, C.P., Cruz, A.S. and Sandbach, J.F. (1983) Prospective cfinical trial of a human tumor cloning system. Cancer Res. 43,1926.
119
Elsevier JIM 05509
Modification of the clonogenic assay for the detection of lymphokine activated killer cell activity * Philip Scuderi ~, Linda Woo ~, Anita S.-F. Chong 2, Rosa Liu t and Sydney E. Salmon t From Arizona Cancer Center, University of Arizona College of Medicine. Tucson. AZ. U.S.A.. and 2 Rush Presbyterian St. Luke's Medical Center. Chicago. IL. U.S.A.
(Received 28 July 1989, revisedreceived30 November 1989. accepted 1 December 1989)
The soft agar clonogenic assay using [3H]thymidine uptake as an endpoint was adapted for the detection of h u m a n LAK activity against the Daudi lymphoma cell line and h u m a n tumor cells obtained by biopsy. Using Daudi cells as the target population the modified agar assay was more sensitive than the conventional 4 h SlCr release assay. Use of a single layer agar assay allowed the assessment of LAK cytotoxic/cytostatic activity against Daudi lymphoma cells after cell to cell contact, while a two layer syste,'n permitted evaluation of the role of soluble mediators in L A K / t a r g e t cell interactions, This study shows, that LAK cells can either kill or inhibit the proliferation of Daudi cells by two mechanisms: one which requires cell-to-cell contact, and a second via soluble mediators. As determined by the use of neutralizing antisera, the soluble factor(s) are not tumor necrosis factor and interferon-,/. Of the nine individual h u m a n tumor samples obtained by biopsy, 89% were sensitive to allogeneic LAK cells when the two populations were admixed. Of these nine tumors 44% were inhibited by LAK-derived soluble factors. The soft agar assay system should serve as a useful tool for determining the sensitivity of h u m a n rumors to L A K cells and for studying the mechanisms of LAK anti-tumor activity. Key words: Clonogenic assay; Lymphokin¢activated ,killer; Cytokine
Introduction The activation of h u m a n peripheral blood leukocytes (PBL) with interleukin-2 (IL-2) gives rise to a phenotypically heterogeneous population of lymphokine activated killer (LAK) cells
Correspondence to: P. Scud¢~'i,Arizona Cancer Center. University of Arizona. 1515 N. Campliell Avenue, Tucson, AZ 85724. U.S.A. * This research was supported by NCI Grants CA21839, CA17094, CA23074and Grant 8277-000000-1-0-YR-9301from the Arizona Disease Control Research Commission. Abbreviations: LAK, lymphokine activated killer; HTCA, human tumor clonogenic assay; TNF, tumor necrosis factor; IFN-y, interferon-y.
(Herberman et al., 1987). In vitro L A K cells are either cytotoxic or cytostatic to established tumor cell lines and to h u m a n tumor cells obtained by surgical excision. In short term isotope release :,ssays the killing of h u m a n tumor cells is not restricted by differences in major histocompatibility complex antigens displayed by the L A K and tumor cell populations and most tumor cells appear to be susceptible to cytolysis ( G r i m m et al., 1982). This non-restricted in vitro anti-tumor activity initially suggested that LAK cell therapy would be applicable to a broad spectrum of human malignancies. To date, however, L A K cell therapy has shown promise for only a fimited number of tumor types (Rosenberg et al., 1985, 1987). The discrepancy between the cytotoxic ac-
0022-1759/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
tivity of LAK cells in vitro and the efficacy of LAK cell therapy suggests that the interaction between effector and target cell populations as measured by the conventional 4 h 51Cr release assay may not be an accurate reflection of LAK cell anti-tumor activity. LAK cells have recently been reported to secrete cytotoxic factors distinct from either tumor necrosis factor or interferon-v (Hersh et al., 1989). The anti-tumor activity of these factors is only detectable in in vitro assay systems such as the soft agar clonogenic assay in which tumor ceils are exposed to soluble mediators for 48 h or more. Thus short term isotope release assays are an ineffective means of measuring anti-tumor activity dependent on LAK derived factors. The soft agar human tumor clonogenic assay (HTCA) is a technique which is widely used to test the activity and clinical utility of chemotherapeutic agents against human tumor ceils obtained by biopsy (Luedke et al., 1984). The HTCA has several distinct advantages over other methods of drug screening, it allows testing to be performed on a small number of tumor cells, the soft agar environment fosters the outgrowth of human t.~mor cells which may not proliferate in fiquid medium, it inhibits the growth of normal fibroblasts, and there is a good correlation between the sensitivity or resistance of tumor cells to a given agent in vitro and the utility or non-utility of that agent in vivo (Von Hoff, 1983). We have developed an approach for use of the soft agar assay for detecting LAK activity against human tumor cells. The system was initially developed using the LAK sensitive Daudi lymphoma cell line and was subsequently used to test the effect of LAK cells on human tumor cells ob= tained by biopsy. Our results suggest that in HTCA human tumor cells are susceptible to either LAK mediated cytolysis or cytostasis after cell to cell contact, in some instances by soluble factors secreted by the LAK cells.
Collection (Rockville, MD) and grown in complete tissue culture medium consisting of RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin and streptomycin. Human tumor cells used in this study which were obtained by biopsy for routine diagnostic purposes after informed consent and were either used immediately or viably frozen in liquid nitrogen until needed. PBL were collected from 24 healthy adult volunteers by venous puncture in heparinized tubes. Mononuclear leukocytes were obtained from heparinized whole blood after separation of Ficoll-Hypaque (Pharmacia, Piscataway, N J). LAK cells were produced by incubating PBL at a density of I x 106/ml in complete medium containing 1000 U / m l IL-2 at 37°C for from 4-7 days. In some experiments LAK cells were irradiated with from 10 to 50 Gray in order to test the effect of LAK proliferation on the assay system.
Reagents Recombinant IL-2 was a gift from Cetus (Emeryville, CA). Recombinant TNF and IFN-'~ were gifts from Genentech (S. San Francisco, CA). Rabbit antiserum specific for either human TNF or IFN-¥ was made by immunizing New Zealand White rabbits with the appropriate recombinant cytokine using established techniques (Scuderi et al., 1987). 51Cr release assay Daudi cells were labelled with 51Cr in the form of sodium chromate and the assays were carded out in round-bottomed 96-weU plates by adding 1 × 104 labelled Daudi cells to each well, followed by the appropriate number of LAK ceils. The plates were spun at 100 X g to facilitate effectortarget cell contact and incubated for four hours at 37°C in a humidified incubator. The percent specific release was calculated using the following formula:
Materials and methods
experimentalrelease-spontaneousrelease × 100 maximumrelease-spontaneousrelease
Cells and culture conditions The human B cell lymphoma cell line Dandi was obtained from the American Type Culture
The experimental release was obtained by incubating effector and target cells at various ratios. Maximum release was produced by adding 1 N
121 HC! to target cells and the spontaneous release was obtained from target cells incubated alone for 4 h at 37°C. All groups were assayed in quadruplicate. Human tumor cells Human tumors were surgically excised, minced into 10 mm 3 sections and placed in sterile hypoosmolar medium consisting of 69 ml of Dulbecco's balanced salt solution supplemented with 8% bovine serum albumin fraction V (Sigma Chemical Co., St. Louis, MO), 20 ml of a 5% solution of polyvinylpyrrolidone (Sigma) in deionized distilled water, 10 ml of 2% methylcellulose (Sigma) in distilled water and 1 ml of a 10% solution of tetrasodiumethylenediaminetetraacetic acid in distilled H20. For each ~ of tumor suspension, 0.5 ml of McCoy's medium containing 3 m g / m l porcine collagenase (Sigma) and 0.3 m g / m l DNase (Sigma) were added. The tumor tissue was subsequently disaggregated by gentle stirring at 37°C for 1 h. The tumor cell suspension was passed through sterile gauze and a 30/~m mesh screen to remove undigested fragments. The tumor cells were washed three times in McCoy's medium containing 10~ fetal calf serum. A single cell suspension was produced by gentle pipetting and ~he viability of each population was assessed by trypan blue due exclusion. Soft agar assay Tumor cells were suspended at a density of 1 × 10 s cells/ml in complete medium. 1 ml of tumor cell suspension was combined with an equal volume of complete medium containing the appropriate number of L A K cells. LAK cells which were stimulated with 1000 U / m l of IL-2 for from 4 - 7 days and were washed three times in Hanks' balanced salt solution (Irvine Scientific, Santa Ana, CA) before being combined with the tumor cells. After a 24 h incubation at 37°C in a humidified incubator 200 /~1 of a 3.5% solution of Sea-Prep 15/45 Agarose (FMC, Rockland, ME) was added. 200/~1 of this cell suspension was then added to each well of a round bottomed 96-well plate. The 0.35% agarose solution containing the cells was solidified by incubating the plate on ice for 30 mln. The assay plates were subsequently incubated at 37°C for 96 h and then pulsed with
3H-TdR by adding 10/~l of a 10/~Ci/ml solution to each well. The plates were then incubated for an additional 48 h. The cells were harvested by floating the assay plates for 2 rain in a 67°C water bath to melt the agarose followed by evacuation of the wells under vacuum and collection of the cells on cellulose acetate filter strips using a multiple automated sample harvester. To ensure that all the cells were collected the wells were repeatedly washed with warmed cae+/Mge+-free phosphatebuffered saline (PBS) supplemented with 37.4 r a g / ! porcine trypsin and 15 m g / l EDTA (Gibco, Grand Island, NY). The harvesting manifold was rinsed in PBS alone between each set of wells. The filter strips containing the immobilized cells were air dried and then immersed in Beckman ReadySole scintillation cocktail (Beckman, Fullerton, CA) and counted in a liquid scintillation counter. Controls included wells containing tumor cells alone and LAK cells alone. The percent inhibition was calculated using the formula: 1
(cffector + target)qam × 100 (effectorcpm) + (target clam)
In the two layer agar system the lower layer was created by solidifying 100 /~! of 0.35% agarose solution in complete rt~edium containing 5 × 104 tumor cells. The upper layer containing the appropriate number of LAK cells was subsequently added and solidified. Incubation times and 3HT d R labelling for the two layer system were the same as used in the single layer assay, except that the upper layer containing LAK cells was removed prior to the addition of isotope.
Results Comparison between L A K activity in the SICr release and soft agar assays For the purposes of comparing the sensitivity of the 51Cr and the single layer agar a~say, PBL were stimulated with IL-2 for 4, 5 and 7 days and then tested on Daudi cells at effector-to-target ratios of from 20 : 1 to 0.08 : 1. Fig. 1 shows that in both the conventional SlCr and the agar assay, 5 day LAK produced the maximum effect. Comparing the two assays, it is clear that the agar assay
122 TABLE I TRITIATED THYMIDINE INCORPORATION IN THE SOFT AGAR HUMAN TUMOR CLONOGENIC ASSAY (HTCA) a LAK : Daudi ratio
Experiment I LAK + Daudi
LAK alone
Experiment 2 LAK + Daudi
LAK alone
Experiment 3 LAK + Daudi
LAK alone
20 : 1
2,908.2 + 925.0
2,354.8 4-372.2
2,598.6 + 362.4
4,253.9 + 493.9
25,549.8 4-2,828.3
32,791.9 + 137.8
10 : 1
3,928.0 + 379.3
934.1 5:47.1
1,588.8 5:152.6
1,038.0 5:219..5
14,075.3 5:1,492.2
17,273.4 + 3,598.4
5 :1
9,809.0 5:4,246.0
463.8 + 66.2
1,101.8 + 212.4
621.4 + 153.4
8,692.3 4-893.5
5,947.9 5:145.9
2.5 : 1
3,030.5 4-1,267.0
190.4 5:69.4
548.5 + 103.7
319.2 5:101.1
4,020.4 + 348.9
1,793.0 5:589.3
1.25 : 1
8,929.0 + 6,159.3
109.4 + 48,2
1,300.6 + 255.4
212.4 + 52,7
9,270.5 :i: 2,181.3
549.3 + 83.1
0.63 : 1
29,603.2 5:1,641.4
85.7 + 39.0
21.905.8 5:2,015.3
101.0 + 21.8
53,281.7 5:2,571.2
383.0 5:327.5
0.31 : 1
36,983.0 + 4,124.2
97.3 + 62.9
58,076.8 + 7,549.9
82.3 + 39.4
133.072.5 + 7,985.0
602.5 :1:245.4
0.16:1
47,870.1 :t: 2,801.1
43.8 :i: 15.4
65,278.9 5:8,068.4
40.0 5:22.6
149,118.3 + 11,508.2
846.5 + 114.6
-
68,881.0 5:3,794.2
31.3 4-15.4
155,799.2 4-10,476.9
2,485.0 4-187.3
0.08 : 1 Daudi alone
47,161.5 5:3.104.6
68,243.4 4-4,091.2
154,415.3 4-2,980.5
a The values shown are the mean of four observations and are expressed as counts/min + standard deviation. LAK cells used in these experiments were produced by incubating human PBL in IL-2 for 5-days.
detects m o r e L A K activity at t h e h i g h e r effectorto-target ratios. T a b l e I s h o w s t h e effect o f a d d i n g t h r e e different L A K p o p u l a t i o n s to D a o d i cells. I n e a c h e x p e r i m e n t L A K cells i n d u c e d a c o n c e n t r a tion d e p e n d e n t decrease in t h y m i d i n e i n c o r p o r a tion. T o test t h e possibility that L A K cells were r e s p o n d i n g to t h e D a u d i cells b y i n c o r p o r a t i n g 3 H - T d R , s o m e L A K cell p r e p a r a t i o n s were irradia t e d with 1 0 - 5 0 G r a y w h i c h criminated t h y m i d i n e i n c o r p o r a t i o n . I n t h e 51Cr a s s a y irradiated L A K r e t a i n e d their cytolytic activity a n d in t h e a g a r a s s a y they p r o d u c e d a n inhibition o f 3 H - T d R c o m p a r a b l e to t h a t i n d u c e d 5 y u r . i ~ a d i a t e d L A K ( d a t a n o t shown).
Optimum length of LAK-Daudi coincubation I n the initial e x p e r i m e n t s 24 h c o i n c u b a t i o n o f L A K a n d D a u d i cells w a s arbitrarily c h o s e n a n d
t h e cells were p l a t e d in a g a r a l o n g with t h e c u l t u r e supernatants. To determine the optimum coincub a t i o n t i m e for L A K a n d D a u d i cells, t h e t w o p o p u l a t i o n s were either c o m b i n e d a n d i m m e d i a t e l y a d d e d to a g a r o r a d m i x e d a n d coincub a t e d for f r o m 3 h to 24 h a n d t h e n p l a t e d in agar. I n addition, t h e effect o f factors w h i c h were prod u c e d d u r i n g t h e c o i n c u b a t i o n period o n t h e D a u d i target cells w a s tested b y w a s h i n g s o m e o f t h e effectors a n d targets after c o i n c u b a t i o n a n d susp e n d i n g t h e m in fresh c o m p l e t e m e d i u m . Fig. 2 s h o w s t h a t t h e o p t i m u m ~ ? i b i t i o n w a s achieved a f t e r a 12 h c o i n c u b a t i o n . T h e s e e x p e r i m e n t s also d e m o n s t r a t e t h a t soluble factors p r o d u c e d d u r i n g t h e c o i n c u b a t i o n d o n o t play a role in t h e inhibition o f 3 H - T d R u p t a k e . I n t h e p r e v i o u s experiment~ t h e a d m i x t u r e of L A K a n d D a u d i at a ratio o f 2 0 : 1 followed i m m e d i a t e l y b y the a d d i t i o n o f t h e cells to a g a r
123 100
O--O 0--0
~-~_
A--A
4- DAY LAK 5 DAY LAK 7 DAY LAK
i1.
\
\\
I
0,01 z
oI 20:1
°'--"-o. \
\
•
\ \ . 10:1
.
. 5:1
.
.
2.S:1
"~0~8~:--~ 1,2S:1 0.63:1 0,S1:1 0.16:1 0.08:t
EFFECTOR : TARGET RATIO
Fig. 1. LAK cells produced by incubating 1 x 106 PBL/ml in complete RPMi medium containing 1000 U / m l recombinant
human IL-2 for the indicated length of time, were admixed with 1 xl04 5lCr-labelled Dandi target cells at the indicated ratios (upper panel). The lower panel shows a soft agar assay which measuresthe inhibitionof 3H-TdR uptake by LAK and Daudi cells at the indicated ratios. 1xl04 Daudi were admixed with LAK and incubated at 37°C for 24 h in complete medium. The cells were subsequently suspended in 0.35% agaros¢ in a 96-weU assay plate. Controls included 3H-TdR uptake by LAK alone at the various ratios used and by Daudi alone. One of two experimentsis shown and each value displayed represents the mean of four replicates+the standard deviation. Where standard deviation bars are not shown they are obscuredby the symbolsize.
were separated in a two layer system. In the two layer system Daudi cells were added to liquid agar which was then solidified on ice and then the LAK cells were over-layered in liquid agar. The upper liquid layer was subsequently solidified on ice and the assay plates were incubated under standard conditions. Under these conditions the two agar layers remain as distinct agar plugs throughout the course of the assay. After an initial 96 h incubation at 37°C the upper agar layer containing the LAK cells was removed under gentle vacuum and the remainir:$ lower layer was pulsed with 3H-TdR. The inhibition in thymidine uptake was equal when cells were either admixed in the same layer of agar or separated in the two layer system (data not shown).
Effect of cytokme-specific antisera on L A K activity Since the two layer HTCA studies s u ~ e s t e d the presence of a diffusible factor(s) which could inhibit thymidine incorporation, cytokine-specific neutralizing antisera were added to the agar to determine whether they could block the diffusible factor activity. Fig. 3 shows that the addition of 10,000 neutralizing umts of rabbit anti-TNF and anti-IFN-y either when the LAK and Daudi were initially admixed or following the twenty-four hour
10o ] E
ao~
20
resulted in a 31% inhibition in thymidine uptake. Under these conditions LAK and Daudi cells were suspended in the agar with few effector and target cells in physical contact with one another. The resultant inhibition suggested that soluble mediators may play some role in the reduced thymidine incorporation. To determine if soluble factors produced after the LAK and Daudi cells were added to a single layer of agar were responsible for the inhibition the two populations were either edmixed and immediately added to agar or they
ol
~ NO WASH P7~ WASH
1i
0 3 6 12 24 TIME EFFECTORS AhD TARGETS COINCUBATEDBEFORE PLATING (hr)
Fig. 2. LAK and Daadi cells were combined at a 20:1 ratio and either plated in agar immediately (0) or coincubated at 37°C for the indicated length of time before the addition of agar. After coincubation, LAK and Daudi were added to the agar either with the conditioned medium (NO WASH) or they were washed in Hanks" balanced salt solution and resuspension in complete medium before plating. One of two experiments is shown and each valut: represents the mean inhibition of 3HTdR uptake of four replicates 4- the standard deviation.
124 OVARIANCARCINOMA
coincubation did n o t effect the inhibition o f thymidine incorporation.
Sensitivity of human tumor cells obtained by biopsy to LAK activity in the single and double layer agar assays T h e u p p e r panel o f Fig. 4 shows that w h e n h u m a n t u m o r cells obtained b y biopsy were admixed with allogeneic L A K for 18 h a n d subsequently a d d e d to the single layer agar system, that eight o f the nine t u m o r cell populations were inhibited. O f the t u m o r types tested only a single l y m p h o m a was refractory to L A K activity in this assay. In contrast to the inhibition o f 3 H - T d R observed w h e n L A K a n d t u m o r were c o i n c u b a t e d a n d then a d d e d to agar, the separation o f effector a n d t u m o r cell p o p u l a t i o n s in two layers (lower panel) resulted in inhibition o f only four o f the nine populations. Interestingly the l y m p h o m a which was uneffected b y coincubation was susceptible to inhibition in the two layer system. E a c h t u m o r cell p o p u l a t i o n was tested with three different allogeneic L A K cell p o p u l a t i o n s a n d in each
Ill
pTA
0':1:t
10080-
~ 60
~,o 20 0
1i1i1i1i NRS
ANTI-TNF ANTI-IFN- T ANTI-TNF
+
ANTI-IFN-7
Fig. 3. LAK and Dandi cells were admixed at a ratio of 20:1 in complete medium alone and in medium containing either 20 /tl of normal rabbit serum or 10 ~tl each of the indicated cytokine-specific antisera. The ceils were incubated 24 h and then added to agar. LAK and Daudi which were initially coincubated alone received either cytokine-spefific antiserum or normal rabbit serum at the time of agar addition. Anti-TNF and anti-IFN-y used in these experiments each contained 1000 neutralizing units of activity/pl. One of four experiments is shown and the values displayed represent the mean of four replicates 4-the standard deviation.
LYMPHOMA E2D I-2-1 MELANOMA SARCOMA
401
00
LAK AND TUMORCELLSADMIXED10:1 RATIO
I I
40]
? [ ~ ADDEDWHEN LAK AND DAUDIWEREADMIXED E22~,t'DDEDWHEN LAK AND DAUDIWERE.ADDEDTO AOAR
l
_
LAK ANDTUMORCELLSSEPARATED10:1 RATIO Fig. 4. Ailogeneic L A K and I x l O 4 human tumor 'oclls obtained by biopsy were admixed at the indicated ratio, in-
cubated for 24 h at 37°C and added to agar (upper panel). The lower panel shows the effect of incubating LAK and human tumor cells separately for 24 h followed by plating the ceils in two separate agar layers. One of three experiments, each using the same tumor cell populations and different allogcneic LAK, is shown and the values displayed represent the mean of four replicate,-.4-the standard deviation.
o f the experiments similar t r e n d s were observed. Six o f the n i n e t u m o r cell p o p u l a t i o n s were tested in a 5tCr assay, however, the s p o n t a n e o u s isotope release was b e t w e e n 40 a n d 56% making the results o f these experiments u n i n t e r p r e t a b l e (data n o t shown).
Effec: of adding cytokine-specific antisera to the two layer agar system with a human lymphoma Sufficient n u m b e r s o f the l y m p h o m a , which was sensitive to b o t h the contact m e d i a t e d a n d soluble effects o f L A K cells, were available to test the role o f T N F a n d I F N - 7 in L A K mediated c y t o t o x i c / c y t o s t a t i c activity. Fig. 5 shows that a
125
6O
]
4o
[ [
20-
ANTI-TNF
I
ANTI--IFN--7
ANTI--TNF + ANTI-IFN-7
Fig. 5. Lymphoma cells were suspended in. complete medium containing 0.35~ agarose at a density of 1 ×105 cells/ml. The rabbit anti-TNF and anti-lFN-? were added at a final concentration of 10,000 U/well, 20 ;tl of normal rabbit serum was used as a control. One of two experiments is shown and the values displayed represent the mean of four observations+ the standard deviation.
suspension of L A K cells in agar containing 10,000 neutralizing units of anti-TNF and anti-IFN-7 either alone or in combination had no effect on the inhibition of 3H-TdR uptake by the lymphoma. In these experiments the upper layer containing immobilized LAK cells was removed at 96 h after which time the lymphoma cells in the lower layer were pulsed with 3H-TdR.
Discussion
This report demonstrates that the soft agar clonogeuic assay can be adapted as a sensitive and reproducible means of detecting L A K activity against human tumor cells. The agar assay has several advantages over the conventional 51Cr release assay. It is more sensitive than the isotope release assay at higher effector-to-target ratios. The enhanced sensitivity of the agar assay is probably due in part to the extended coincubation time within which LAK and tumor cells are in physical contact. Additional sensitivity may also occur because the agar assay detects cytotoxicity mediated by both cell contact and soluble factors. Thus the second advantage of the agar assay is that it can be used, in the two laver format, to distinguish between direct cell-mediated and secreted factor-
mediated cytotoxicity. LAK cell populations can secrete a variety of cytotoxic cytokines including T N F and IFN-y and the release of both T N F and IFN-y can be stimulated by human tumor cells (Chong et al., 1989a,b). L A K cells are also capable of secreting one or more unique cytotoxic cytokines distinct from T N F and IFN-'r (Hersh et al., 1989). The third advantage of the assay system is that the soft agar environment allows the screening of fresh human tumor biopsies which may preferentially grow when immob'flized in agar (Hamburger et al., 1978). Both the single layer and double layer agar systems described in this report should be adaptable for the study of other effector populations. In the present report heterogenous L A K cells were studied. The 3H-TdR uptake used as an endpoim in these assays was suitable for measuring tub, or cell proliferation because L A K deprived of IL-2 failed to proliferate and incorporate thymidine in our system. The introduction of IL-2 into the agar assay haduced LAK proliferation which was unpredictable and often exceeded that of tumor cell populations (data not shown). However, in related experiments we have determined that irradiation of LAK cells with up to 5000 rads can inhibit L A K proliferation but still permit LAK-induced inhibition of target cell 3H-TdR incorporation. Thus one limitation of the agar system is that the effector population must either not proliferate o~" they must take up thymidine at levels well below that of the tumor target cell population. This requirement may be met by macrophages and monocytes, but may not be possible when using T cells as the effector population. The experiments reposed here suggest that both the single and double layer agar system may be a useful tool for studying L A K cell mediated cytolysis. Our findings are consistent with pubEshed accounts of L A K activity measured in single layer agar assays using colony counting as an endpoint (Bradley et al., 1985; Klvin-Nelemans et al., 1989). Further, our system which uses 3H-TdR vptake as an end point may be adaptable for the study of cytotoxic and cytostatic activity produced by other cell types. The assays described here may also provide a means of studying both contact and factor mediated antitumor activity produced by a single effector cell population.
126 Referenee~ Bradley, E.C., Catino, J.J., Issell, B.F., Pniesz, B., Huslad, J.M., Dalton, T., Allegretta, M. and Mier, J. (1985) Cell-mediated inhibition of tumor colony formation in agarose by resting and interleukin 2-stimulated human iymphocytes. Cancer Res. 45,1464. Chong, A.S.-F., Aleksijevic, A., Scoderi, P., Hersh, E.M. and Grime.s, W.J. (1989a) Phenotypic and functional analysis of LAK cell clones: Ability of CD3 + LAK cell clones to produce IFN-gamma and tumor necrosis factor upon stimulation with tumor targets. Cancer Immunol. Immunotber. 29, 270. Chong, A.S.-F., Scuderi, I~., Grimes, W.J. and Hersh, E.M. (1989b) Tumor targets stimulate IL-2 activated killer cells to produce IFN-y and tumor necrosis factor. J. Immunol. 142. 2133. Grimm, E.A., Mazumder, A,, Zhong, A,Z. and Rosenberg, S.A. (1982) Lymphokine-activated killer cell phenomenon lysis of natural killer-resistant fresh solid tumor cells by interleuldn 2-activated autologoas human peripheral blood iymphocytes. J. Exp. Med. 155,1823. Hamburger, A., Salmon, S.E., Kim, M., Trent, J.M., Soehnlen, B.J., Aiberts, D.S. and Schmidt, H.J. (1978) Direct cloning of human ovarian carcinoma cells in agar. Cancer Res. 38, 3438. Herberman, R.B., Hiserodt, J., Vujanovic, N , Balch, C., Lotzova, E., Bolhvis, R., Golub, S., Lanier, L.L., Philfips, J.H., Riccardi, C., Ritz, J., Santoni, A., Sclunidt, R.E. and Uchidm. A. (1987) Lzmphokine-aetivated killer cell activity. Immunol. Today 8,178. Harsh, E.M., Scuderi, P., Grimes, W.J., Chong, .~ ' .F., Brailey, J.L., Gschwind, C.R. and Salmon, S.E. (l:~og) Cytotoxic and cytostatic activity of lymphokine activated killer
(LAK) cell supematants. Cancer lmmunol, lmmunother. 30, 65. KlvinoNelemans, H.C., Van der Harst, D , Van den Burgh, J.F., Van Luxemburg, S. and Brand, A. (1989) Comparison between clonogenic and cytotoxic assays for measuring LAK cell activity. Intern. J. Cell Cloning 7, 232. Luedke, D.W., Carey, F.J., Krishan, A., Chce, D., Chang, B., Franco, R., Niell, H.B., Jones, M.E., Kenady, D.E. and Zirvi, K. (1984) Interlaboratory reproducibifity of the human tumor colony forming assay (HTCA): The Southeastern Center Study G~oup (SECSG) experience. In: S.E. Salmon and J.M. Trent (Eds.), Human Tumor Cloning. Grune and Stratton, Orlando, FL, p. 1245. Rosenberg, S.A., Lotze, M.T., Muul, L.M., Leitman, S., Chang, A.E., Ettinghausen, S.E. Matory, Y.L., Skibber, J.M., Shiloni, J.M., Vetto, J.T., Seipp, C.A., Simpson, C. and Reichert, C.M. (1985) Observations on the systemic administration of autologous lymphnkine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. New Engi. £ Med. 313,1485. Rosenberg, S.A., Lotze, M.T., Muul, L.M., Chang, A.F., Auis, F.P., Leitman, S., Linehan, W.M,, Robertson, C.N., Lee, R.E., Rubin, J.T., Seipp, C.A,, Simpson, C.G. and White, D.E. (1987) A progress report on the treatment of 157 patients with advanced cancer using iymhokine-aetivated killer cells and interleukin-2 or high dose interleukin-2 alone. New Engl. J. Med. 316, 889. Scuderi, P., Sterling, K.E., Raitano, A.B., Grogan, T.M, and Rippe, R.A. (1987) Recombinant gamma interferon stimulates the production of tumor necrosis factor in vitro. J. Interferon Res. 7,155. Von Hoff, D.D., Clark, G.M., Stogdill, B.J., Sarnsdy, M.F., O'Brien, M.T., Casper, J.T., Mattox, D.E., Page, C.P., Cruz, A.S. and Sandbach, J.F. (1983) Prospective cfinical trial of a human tumor cloning system. Cancer Res. 43,1926.