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Production of lymphokines affecting tumor cells by T-T hybridomas
CELLULAR
IMMUNOLOGY
93, 54 l-548 ( 1985)
Production of Lymphokines Affecting Tumor Cells by T-T Hybridomas’ MARION
C. COHEN AND MELINDA
LAZARUS
Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut 06032 Received December 28, 1984; accepted March 5, 1985 T-Cell hybridomas were constructedby fusing BW5 147,an AKR lymphoma, with concanavalin A-stimulated murine splenic lymphocytes. The hybrids which were formed were studied for their ability to produce a lymphokine which inhibits tumor cell migration (TMIF) as well as macrophage migration (MIF) using in vitro assays.Clones were identified which affected tumor cell motility without exerting similar effects on murine macrophages, although the opposite effect was not observed. Although noncoordinate production of these factors cannot be unequivocally established, these results demonstrate that clones can be constructed that preferentially secrete TMIF. In these experiments, we also tested the supematants for another lymphokine effect on tumor cells; namely, the ability to inhibit tumor cell binding to endothelial monolayers. A number of clones were identified that lacked TMIF activity, but could inhibit the tumor cell-endothelial interaction, suggestingthe possibility that these effects may be due to separate mediators. 0 1985 Academic Press, Inc.
INTRODUCTION
The possibility that products of the immune system could influence host defense against tumors has been suggested by a variety of clinical and experimental observations including the discovery of a wide variety of tumor-specific antigens and the development of a number of animal models in which immunization against such antigens modified the behavior of transplanted, induced, or spontaneous neoplasms (reviewed in (1)). It is generally believed that tumor immunity is a manifestation of cell-mediated immunity (reviewed in (2)) but there is also the possibility that humoral antibodies may play a role in controlling tumor growth by destroying neoplastic cells in the presence of complement. While most studies have focused on mechanisms of tumor cell cytolysis, it is equally likely that products of the immune system might guard the host from either invasiveness or metastasis of tumor cells without direct cell killing. Based on these considerations, we investigated the ability of lymphokines to influence various functional properties of tumor cells. By analogy with mechanisms by which inflammatory cell movement may be modified during cell-mediated immune reactions, we have found that tumor cell movement may also be affected by lymphokines derived from antigen- or mitogenstimulated lymphocytes or long-term lymphoblastoid cell lines in migration assays ’ Supported by NIH Grant CA-323 19.
ooog-8749185S3.00 Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
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involving experimental tumors in ascitic form (3-5). We found that these lymphokinecontaining supernatants were capable of inhibiting the migration of ascitic lines of tumor cells using both capillary tube and agarose microdroplet assays originally developed for studying inflammatory cell movement (6). These results have been extended to studies of solid neoplasms of experimental origin as well as primary human neoplasms (7, 8). Physicochemical characterization studies have suggested that the factor which inhibits tumor cell migration is separable from factors which inhibit inflammatory cell migration (4, 5). Attempts to find pure sources of a single lymphokine have not met with much success.Even hybridomas tend to produce multiple lymphokines (9-l 1). However, the number of mediators produced is often fewer than that generated by stimulated normal lymphocytes or long-term lymphoblastoid lines. Thus, it is possible to determine the identity or nonidentity of two lymphokine activities by obtaining clones that produce one or the other, but not both. In addition, hybridomas serve as convenient sources of mediator for ultimate separation and purification. In this study we have used hybridoma methodology to develop tumor migration inhibition factor (TMIF)-secreting lines which are without detectable migration inhibition factor (MIF) activity as well as lines which appear to secreteboth factors. In addition, we have used these lines to study another noncytotoxic lymphokine effect on tumor cells, namely, the ability to interfere with their binding to endothelial monolayers. MATERIALS
AND METHODS
Animals. AKR female mice, 4-5 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, Maine) for preparation of spleen cell cultures. BALB/c female mice, 4-5 weeks old (Jackson Laboratory) were used as a source of peritoneal exudate cells. P8 15 mastocytoma was maintained in DBA/2 female mice, 4-5 weeks old (Jackson Laboratory). Preparation of spleen cells for fusion. Cell suspensionswere obtained from normal AKR mice by forcing spleens through stainless steel net (150 mesh) with a rubber policeman. After two washings with Hanks’ balanced salt solution (HBSS), the cells were counted, viability determined, and the cells were resuspended in RPM1 1640 medium (GIBCO, Grand Island, N.Y.) supplemented with antibiotics and 10% fetal calf serum (HyClone, Logan, Utah) at a concentration of 1 X lo7 cells/ml. Concanavalin A (Con A) (Miles-Yeda, Rehovot, Israel) was added to a concentration of 5 &ml of cell suspension. The cultures were incubated at 37°C in an atmosphere of 5% C02-95% air for 18 hr. Preparation of T-cell hybrids. Spleen cells were collected following incubation, centrifuged at 200g for 5 min and resuspended in Dulbecco’s minimum essential medium (MEM; 4500 mg glucose/liter) (GIBCO). The cells were counted and viability determined. The cells were mixed with BW5147 (a gift from Dr. Nancy Ruddle) in a spleen lymphoma ratio of 4: 1 and the fusion was performed according to the method of Beezley and Ruddle (12) except that Koch-Light PEG 1000 (Research Products International, Mount Prospect, Ill.) was used as the fusing agent. Cells were dispensed into a 96-well tissue culture plate (Costar, Cambridge, Mass.) at a concentration of 3 X lo5 BW5147 lymphoma cells/ml and 120 pgl of this
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suspension was placed in each well of 96-well tissue culture plates (Costar). The plates were incubated at 8% C02-95% air at 37°C. Hybrids appeared at IO-14 days after fusion. Lymphokine assays. MIF was assayed using the agarose microdroplet assay and TMIF was assayedusing both the capillary tube and agarose microdroplet assaysas described previously (6). Briefly, murine peritoneal macrophages were induced by injection of 2 ml sterile light mineral oil 4 days prior to assay.The mice were killed by exsanguination and their peritoneal cavities washed with HBSS. The cells were washed twice and the final pellet was resuspended in 3 vol of a mixture of RPM1 1640 containing 0.2% SeaPlaque agarose (Marine Colloid, Rockland, Maine) and 10% heat-inactivated fetal calf serum. A l-p1 droplet of the cell suspension was placed in the center of a 96-well tissue culture plate (Falcon Microtest III, Oxnard, Calif.) with a 50-~1 Hamilton syringe equipped with an automatic dispenser and a 22-gauge blunt-tipped needle. The droplets were allowed to gel at 4°C for 12 min. Chilled test medium was gently added (100 &well). Each sample was assayed in quadruplicate. The plates were incubated at 37°C for 18 hr in a humidified atmosphere of 5% CO*-95% air. The migration was quantified on an inverted microscope equipped with a 0.5-mm* reticule (Edmund Scientific, Barrington, N.J.). Five measurements were made on each droplet: the radius of the original droplet and the distance from the edge of the droplet to the periphery of the migration front at 4 points 90” from each other and tangential to the migration fronts. The average of the migration distances of a particular sample plus the average radius of the droplets in that sample were used to calculate the total area of the droplet plus the migration. The area of the original droplet was subtracted from the total area, yielding the migration area. Percentage inhibition of migration for each sample was calculated as follows: area in experimental supernatants x 100. area in control supernatants > P8 15 mastocytoma cells were maintained by serial intraperitoneal passageof 0.1 ml ascites at weekly intervals. The agarose microdroplet assay as described above was used in some assays,with tumor cells substituted for peritoneal exudate cells. At other times the capillary tube method was used. We have shown that these two assays may be used interchangably (6). Capillary tubes were packed with tumor cells suspended in RPM1 1640 supplemented with antibiotics and 10% fetal calf serum to a total of 15X packed cell volume. A volume of 1.5 ml of the cell suspension was used to pack 18 capillary tubes (34507 Kimble, VWR, Boston, Mass.). The cell suspension was drawn up into tubes by reducing the ambient pressure by means of a vacuum pump. The tubes were cut below the cell-fluid interface and two capillaries were anchored in silicone greaseon a cover slip placed in a Sykes-Moore chamber (Bellco, Vineland, N.J.). Each chamber was filled with 1 ml of experimental or control test medium and incubated at 37°C. After 18-24 hr, the area of migration of cells in control and experimental media was determined by tracing the periphery of a magnified projection of the migrating cells on an Epoi LP-6 profile projector (Ehrenreich Photo Optical Industries, Inc., Garden City, N.Y.) and measuring it with a planimeter (LASICO, Los Angeles, Calif.). Percentage
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migration inhibition based on the average of four replicate samples was calculated as follows: % inhibition = 1 (
area in experimental supernatants x 100. area in control supernatants 1
In both migration inhibition assays, viability was measured by trypan blue exclusion and radiochromium release as previously described (3-6). In addition, both assaysshow reversibility; after continued incubation (48 to 72 hr) the migrationinhibited preparations regain the capacity for migration and become indistinguishable from controls. Tumor cell-endothelial cell binding assay. To study tumor cell binding to endothelial monolayers, tumor cell suspensions were obtained as described above. Endothelial monolayers were prepared from bovine pulmonary artery endothelial cells as described by Picciano et al. (13). Their identity as endothelial cells was confirmed by the detection of factor VIII antigen by indirect immunofluorescence (14). The tumor cells were labeled with “Cr by standard techniques (15). Tumor cells were resuspended at 5 X lo4 cells/ml in experimental or control supematant. Culture medium was aspirated from the wells of 12-well tissue culture plates (Falcon Plastics) and a l-ml suspension of tumor cells was added to each well. The plates were incubated at 37°C in a humidified 5% C02-95% air atmosphere for 60 min. Each culture plate contained samples in experimental and control media, thus providing an internal control for the consistency of the separation procedure. Upon completion of the incubation, nonadherent cells were removed by pipetting and the monolayers were washed four times with RPM1 1640 medium containing 2% FCS. After the last wash, the contents of each well, consisting of adherent labeled tumors and unlabeled endothelium, was solubilized by adding 1 ml of 0.5 N NaOH and the level of radioactivity was determined in a Beckman gamma counter. This procedure, which is a standard assayfor tumor binding ( 16), has good reproducibility, as seen in the very small standard errors in Table 2 (see below). RESULTS Initial screening for TMIF was performed using the agarose microdroplet assay which requires only small quantities of supernatant. Culture supematants from 36 hybridomas were tested. Cells from samples which showed marked TMIF activity were expanded to larger volumes. To prepare supernatants for further testing, l-2 X lo6 cells were placed in culture for 24 hr. The supernatants from these cultures were harvested and tested for both TMIF and MIF activity. In addition, supernatants were prepared from the myeloma fusion partner, BW5 147, and assayedfor inhibitory activity. The results in Table 1 show that supernatants prepared from BW5147 did not inhibit the migration of either tumor cells or murine PEC. Of the four other uncloned hybrids for which data is included, two (lB5 and 1ClO) could inhibit both tumor and macrophage migration. One hybrid (lB7) was negative for both activities. Supematants prepared from the fourth hybrid (3F4) could inhibit tumor cell but not macrophage migration. We prepared a number of clones from 3F4. The data derived from four of them are shown in Table 1. One cloned sample did not continue to release TMIF. Three
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p815”
PEC”
BW5 147 (fusion partner) IB5 IB7 IClO 3F4 2B6b 2C4’ 2C8’ 2C9’
0.2 ic 5.5 25.0 f 1.8 -5.3 f 4.7 20.2 f 4.6 38.2 f 3.9 2.9 zk 4.1 36.1 + 4.5 35.5 f 3.4 31.4 + 4.1
2.9 + 8.8 20.8 f 9.9 -9.5 f 10.2 24.8 f 6.9 15.5 + 7.0 nd’ 8.6 * 7.9 17.0 z!T 7.7 17.9 f 7.6
‘Values > 20% are considered to represent significant inhibition (6). Of the subclones, 2C4 has no activity against PEC, whereas 2C8 and 2C9 have marginal activity. All three, however, are active against P815. bCloned from 3F4. ’ nd = not done.
other clones behaved in the same manner as the hybrid from which they were derived. Supernatants from 2C4, 2C8, and 2C9 continued to releaseTMIF but only 2C4 did not affect macrophage migration. Clones 2C8 and 2C9 appeared to release marginal quantities of macrophage MIF. This pattern has continued. In all migration inhibition assays,viability was AO% with no difference between experimental and control preparations. Migration-inhibited preparations showed reversibility of effect; continued incubation for an additional 48-72 hr led to recovery of the capacity for migration. We next attempted to determine whether these supernatants exerted other noncytotoxic effectson tumor cell behavior. We have recently found that lymphokineTABLE 2 Effect of Hybridoma Supernatants on Tumor Cell Adherence to Endothelial Monolayers % Adherence (mean + SE; n = 4)b Supernatant Complete medium Unstimulated normal lymphocytes BW5 147 1B7 2B6 2C8
TMIF”
Expt I
Expt 2
Expt 3
72.3 f 0.9
75.1 * 4.0
16.4 f 4.4
74.8 f 50.6 f 39.4 + 47.0 + 54.0 f
72.9 5 2.8 54.7 f 3.6 (27.2) nd nd 46.3 + 4.2 (38.3)
69.3 f 58.5 + 52.3 k 63.9 f
1.6 1.2 (30.0) 1.0 (45.5) 3.7 (35.0) 2.0 (25.3)
nd 2.1 (9.3) 2.7 (23.4) 3.8 (31.5) 1.5 (16.4)
’ For comparison, the presence or absence of TMIF is indicated for each supematant. b% Adherence determined by radiolabel assaydescribed in the text. Figures in parenthesesrepresent % inhibition of adherence relative to medium control.
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containing supernatants derived from a lymphoblastoid long-term cell line as well as supernatants prepared from Con A-stimulated murine spleen cells known to contain TMIF were capable of suppressing the attachment of tumor cells to endothelial monolayers (17). It was, therefore, of interest to see if supematants prepared from hybridoma sources of which one member of the pair was a Con Aactivated spleen cell could behave in a similar fashion. The results in Table 2 indicate that hybridoma-derived supernatants could decreasetumor cell adherence. However, this did not correlate with the presence of TMIF. Furthermore, the fusion partner, BW5147, was also capable of modifying attachment of tumor cells to endothelial monolayers. DISCUSSION The ability to use hybridoma technology to produce clones of cells derived from one cell is of great potential in determining whether effector lymphokine molecules are responsible for more than one activity. A number of lines have been prepared which appear to secrete either one or only a very limited number of molecules (9-l 1, 18, 19). We have fused Con A-activated murine spleen cells, which normally produce migration inhibitory factors, with BW5 147 to produce a hybridoma which secretes a product capable of modifying the migration of neoplastic but not inflammatory cells as well as a number of other hybridomas that secrete products capable of inhibiting both. The ability to separatethese two lymphokine effectsis more than just an academic exercise. First, the question of whether one cell is limited to the production of one lymphokine has not yet been answered, since it is known that some lymphokines or cytokines can have multiple activities (20-23). Second, while it would be desirable to use migration inhibitory factors to prevent neoplastic cells from spreading throughout the organism, their use would be limited if inflammatory cells were affected as well. Our results indicate that it is possible to prepare supematants with TMIF activity devoid of MIF activity. Whether this indicates that these are two distinct molecular speciesis not clear because we have not been able to obtain lines of cells with MIF but not TMIF activity. Such a reciprocal finding would unequivocally exclude differences in assay sensitivity as a cause of the observed results. However, we have already found such reciprocal dissociations in our physicochemical characterization studies (3-5). Thus, incubation with fucose and rhamnose abolishes MIF but not TMIF activity, while conversely, diisopropyl fluorophosphate (DFP) inhibits TMIF but not MIF. Under appropriate conditions of culture, some murine strains produce TMIF in the absence of MIF, whereas guinea pig cells produce MIF but not TMIF. More evidence that the results reported here are not due to differences in assay sensitivity is provided by the observation that both activities are of approximately the same magnitude in activated normal lymphocyte cultures. Also, we never saw MIF activity reappear in the clones derived from 3F4, which would be expected if lack of MIF activity were due simply to different amounts of factors secreted. Finally, the previously reported size differences between TMIF and MIF provide further evidence for their nonidentity. However, it should be noted that absolute proof that TMIF can be produced in the absenceof MIF must await the development
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of a sensitive radioimmunoassay for both. Moreover, it is also possible that TMIF could be a subunit of the larger MIF molecule; i.e., the entire molecule is not necessaryfor an effect on tumor cells. There is evidence (24) that MIF is composed of two subunits, one of which is required for attachment to the target cell while the other exerts the observed biological effect. The latter is found in the same molecular weight range as TMIF and it is tempting to speculate that they might be identical or related. We have recently extended our studies of noncytotoxic lymphokine effects on tumor cells and have found a factor which causesdecreasedbinding of tumor cells to endothelial cell monolayers (17). This factor is found in supernatants known to contain TMIF activity, and in preliminary studies, appears to exert its effect on the tumor cells themselves. We found the activity in all the hybridoma supernatants that we tested, including supernatants prepared from the BW5 147 lymphoma line used for fusion. In contrast, we, and others, have never found migration inhibitory activities present in supematants prepared from this cell line (Table 1; Ref. (9)). Thus, it is possible the migration inhibitory activity and the binding inhibitory activities are distinct. Further studies will be required to confirm whether these activities are, in fact, due to different factors. It is clear that TMIF production is not merely a property of all growing cells, since the fusion partner, BW5147, was itself unable to produce TMIF. Also, we have previously reported that antigen-activated (3) or mitogen-activated (4) lymphocytes, but not unstimulated ones, produce TMIF. The situation for tumor-cell binding is less clear since, as indicated above, BW5 147 does produce a factor that inhibits tumor cell binding to endothelium. However, as seenin Table 2, nonactivated normal lymphocytes do not. Although antigen-activated murine lymphocytes also produce this binding inhibitory activity, antigen-activated guinea pig lymphocytes do not (unpublished observation), providing further evidence that this activity is not a nonspecific effect of cell culture supematants. Our present results demonstrate that T-cell hybridomas can be generated which constitutively produce factors which affect the behavior of neoplasms but are not cytotoxic for these cells. These cell lines represent continuous sources of mediators which have the potential for being relatively free of contamination by mediators with similar biologic behavior for other cell types. They therefore provide a convenient source of mediator for both purification and studies of potential therapeutic effect in viva REFERENCES 1. Ristow, S., and McKhann, C., In “Mechanisms of Tumor Immunity” (I. Green, S. Cohen, and R. T. McCluskey, Eds.). Wiley, New York, 1977. 2. Herberman, R. B., and Holden, H. T., A& Cancer Rex 27, 305, 1978. 3. Cohen, M. C., Zeschke, R., Bigazzi, I’. E., Yoshida, T., and Cohen, S., J. Immunol. 114, 1641, 1975. 4. Cohen, M. C., Goss, A., Yoshida, T., and Cohen, S., J. Immunol. 121, 840, 1978. 5. Cohen, M. C., Cancer Rex 42, 2135, 1982. 6. Adelman, N., Hasson, M., Masih, N., and Cohen, M. C., J. Immunol. Methods 34, 235, 1980. 7. Donskoy, M., Forouhar, F., and Cohen, M. C., Cancer Rex 44, 3870, 1984. 8. Cohen, M. C., Forouhar, F., Donskoy, M., and Cohen, S., Clin. Immunol. Immunopathol. 34, 94, 1985. 9. Jones, C. M., Braatz, J. A., and Herberman, R. B., Nature (London) 291, 502, 1981.
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10. Schreiber, R. D., Altman, A., and Katz, D. H., J. Exp. Med. 156, 677, 1982. 11. Erickson, K. L., Cicurel, L., Gruys, E., and Fidler, I. J., CeN. Immunol. 72, 195, 1982. 12. Beezley, B., and Ruddle, N. H., J. Immunol. Methods 52, 269, 1982. 13. Picciano, P. T., Johnson, B., Walenga, R. W., Donovan, M., Borman, B. J., Douglas, W. H. J., and Kreutzer, D. L., Exp. Cell. Rex 151, 134, 1984. 14. Ryan, U. S., Clements, E., Habliston, D., and Ryan, J. W., Tissue Cell 10, 535, 1978. 15. Munger, W., and Lindquist, R., In “Investigation of Cell-Mediated Immunity” (T. Yoshida, Ed.). Churchill Livingstone, Edinburgh, in press. 16. Kramer, R. H., and Nicolson, G. L. Proc. Natl. Acad. Sci. USA 76, 5704, 1979. 17. Cohen, M. C., Mecley, M., Antonia, S. J., and Picciano, P. T., submitted for publication. 18. Higuchi, M., Asada, M., Kobayashi, Y., and Osawa, T., Cell. Immunol. 78, 257, 1983. 19. Kaltmann, B., Gemsa, D., Hultner, L., Kees, U., Marcucci, F., and Krammer, P. H., In “Interleukins, Lymphokines and Cytokines” (J. J. Oppenheim and S. Cohen, Eds.). Academic Press,New York, 1983. 20. Hefeneider, S. H., Conlon, P. J., Henney, C. S., and Gillis, S., J. Immunol. 130, 222, 1983. 21. Atkins, E., J. Infect. Dis. 149, 339, 1984. 22. Steeg, P. S., Moore, R. N., Johnson, H. M., and Oppenheim, J. J., J. Exp. Med. 156, 1780, 1982. 23. Wong, G. H. W., Clark-Lewis, I., McKimm-Breschkin, J. L., Harris, A. W., and Schrader, J. W., J. Immunol. 131, 788, 1983. 24. Possanza,G., Cohen, M. C., Yoshida, T., and Cohen, S., Science (Washington, D.C.) 205, 300, 1979.
IMMUNOLOGY
93, 54 l-548 ( 1985)
Production of Lymphokines Affecting Tumor Cells by T-T Hybridomas’ MARION
C. COHEN AND MELINDA
LAZARUS
Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut 06032 Received December 28, 1984; accepted March 5, 1985 T-Cell hybridomas were constructedby fusing BW5 147,an AKR lymphoma, with concanavalin A-stimulated murine splenic lymphocytes. The hybrids which were formed were studied for their ability to produce a lymphokine which inhibits tumor cell migration (TMIF) as well as macrophage migration (MIF) using in vitro assays.Clones were identified which affected tumor cell motility without exerting similar effects on murine macrophages, although the opposite effect was not observed. Although noncoordinate production of these factors cannot be unequivocally established, these results demonstrate that clones can be constructed that preferentially secrete TMIF. In these experiments, we also tested the supematants for another lymphokine effect on tumor cells; namely, the ability to inhibit tumor cell binding to endothelial monolayers. A number of clones were identified that lacked TMIF activity, but could inhibit the tumor cell-endothelial interaction, suggestingthe possibility that these effects may be due to separate mediators. 0 1985 Academic Press, Inc.
INTRODUCTION
The possibility that products of the immune system could influence host defense against tumors has been suggested by a variety of clinical and experimental observations including the discovery of a wide variety of tumor-specific antigens and the development of a number of animal models in which immunization against such antigens modified the behavior of transplanted, induced, or spontaneous neoplasms (reviewed in (1)). It is generally believed that tumor immunity is a manifestation of cell-mediated immunity (reviewed in (2)) but there is also the possibility that humoral antibodies may play a role in controlling tumor growth by destroying neoplastic cells in the presence of complement. While most studies have focused on mechanisms of tumor cell cytolysis, it is equally likely that products of the immune system might guard the host from either invasiveness or metastasis of tumor cells without direct cell killing. Based on these considerations, we investigated the ability of lymphokines to influence various functional properties of tumor cells. By analogy with mechanisms by which inflammatory cell movement may be modified during cell-mediated immune reactions, we have found that tumor cell movement may also be affected by lymphokines derived from antigen- or mitogenstimulated lymphocytes or long-term lymphoblastoid cell lines in migration assays ’ Supported by NIH Grant CA-323 19.
ooog-8749185S3.00 Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
542
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involving experimental tumors in ascitic form (3-5). We found that these lymphokinecontaining supernatants were capable of inhibiting the migration of ascitic lines of tumor cells using both capillary tube and agarose microdroplet assays originally developed for studying inflammatory cell movement (6). These results have been extended to studies of solid neoplasms of experimental origin as well as primary human neoplasms (7, 8). Physicochemical characterization studies have suggested that the factor which inhibits tumor cell migration is separable from factors which inhibit inflammatory cell migration (4, 5). Attempts to find pure sources of a single lymphokine have not met with much success.Even hybridomas tend to produce multiple lymphokines (9-l 1). However, the number of mediators produced is often fewer than that generated by stimulated normal lymphocytes or long-term lymphoblastoid lines. Thus, it is possible to determine the identity or nonidentity of two lymphokine activities by obtaining clones that produce one or the other, but not both. In addition, hybridomas serve as convenient sources of mediator for ultimate separation and purification. In this study we have used hybridoma methodology to develop tumor migration inhibition factor (TMIF)-secreting lines which are without detectable migration inhibition factor (MIF) activity as well as lines which appear to secreteboth factors. In addition, we have used these lines to study another noncytotoxic lymphokine effect on tumor cells, namely, the ability to interfere with their binding to endothelial monolayers. MATERIALS
AND METHODS
Animals. AKR female mice, 4-5 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, Maine) for preparation of spleen cell cultures. BALB/c female mice, 4-5 weeks old (Jackson Laboratory) were used as a source of peritoneal exudate cells. P8 15 mastocytoma was maintained in DBA/2 female mice, 4-5 weeks old (Jackson Laboratory). Preparation of spleen cells for fusion. Cell suspensionswere obtained from normal AKR mice by forcing spleens through stainless steel net (150 mesh) with a rubber policeman. After two washings with Hanks’ balanced salt solution (HBSS), the cells were counted, viability determined, and the cells were resuspended in RPM1 1640 medium (GIBCO, Grand Island, N.Y.) supplemented with antibiotics and 10% fetal calf serum (HyClone, Logan, Utah) at a concentration of 1 X lo7 cells/ml. Concanavalin A (Con A) (Miles-Yeda, Rehovot, Israel) was added to a concentration of 5 &ml of cell suspension. The cultures were incubated at 37°C in an atmosphere of 5% C02-95% air for 18 hr. Preparation of T-cell hybrids. Spleen cells were collected following incubation, centrifuged at 200g for 5 min and resuspended in Dulbecco’s minimum essential medium (MEM; 4500 mg glucose/liter) (GIBCO). The cells were counted and viability determined. The cells were mixed with BW5147 (a gift from Dr. Nancy Ruddle) in a spleen lymphoma ratio of 4: 1 and the fusion was performed according to the method of Beezley and Ruddle (12) except that Koch-Light PEG 1000 (Research Products International, Mount Prospect, Ill.) was used as the fusing agent. Cells were dispensed into a 96-well tissue culture plate (Costar, Cambridge, Mass.) at a concentration of 3 X lo5 BW5147 lymphoma cells/ml and 120 pgl of this
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suspension was placed in each well of 96-well tissue culture plates (Costar). The plates were incubated at 8% C02-95% air at 37°C. Hybrids appeared at IO-14 days after fusion. Lymphokine assays. MIF was assayed using the agarose microdroplet assay and TMIF was assayedusing both the capillary tube and agarose microdroplet assaysas described previously (6). Briefly, murine peritoneal macrophages were induced by injection of 2 ml sterile light mineral oil 4 days prior to assay.The mice were killed by exsanguination and their peritoneal cavities washed with HBSS. The cells were washed twice and the final pellet was resuspended in 3 vol of a mixture of RPM1 1640 containing 0.2% SeaPlaque agarose (Marine Colloid, Rockland, Maine) and 10% heat-inactivated fetal calf serum. A l-p1 droplet of the cell suspension was placed in the center of a 96-well tissue culture plate (Falcon Microtest III, Oxnard, Calif.) with a 50-~1 Hamilton syringe equipped with an automatic dispenser and a 22-gauge blunt-tipped needle. The droplets were allowed to gel at 4°C for 12 min. Chilled test medium was gently added (100 &well). Each sample was assayed in quadruplicate. The plates were incubated at 37°C for 18 hr in a humidified atmosphere of 5% CO*-95% air. The migration was quantified on an inverted microscope equipped with a 0.5-mm* reticule (Edmund Scientific, Barrington, N.J.). Five measurements were made on each droplet: the radius of the original droplet and the distance from the edge of the droplet to the periphery of the migration front at 4 points 90” from each other and tangential to the migration fronts. The average of the migration distances of a particular sample plus the average radius of the droplets in that sample were used to calculate the total area of the droplet plus the migration. The area of the original droplet was subtracted from the total area, yielding the migration area. Percentage inhibition of migration for each sample was calculated as follows: area in experimental supernatants x 100. area in control supernatants > P8 15 mastocytoma cells were maintained by serial intraperitoneal passageof 0.1 ml ascites at weekly intervals. The agarose microdroplet assay as described above was used in some assays,with tumor cells substituted for peritoneal exudate cells. At other times the capillary tube method was used. We have shown that these two assays may be used interchangably (6). Capillary tubes were packed with tumor cells suspended in RPM1 1640 supplemented with antibiotics and 10% fetal calf serum to a total of 15X packed cell volume. A volume of 1.5 ml of the cell suspension was used to pack 18 capillary tubes (34507 Kimble, VWR, Boston, Mass.). The cell suspension was drawn up into tubes by reducing the ambient pressure by means of a vacuum pump. The tubes were cut below the cell-fluid interface and two capillaries were anchored in silicone greaseon a cover slip placed in a Sykes-Moore chamber (Bellco, Vineland, N.J.). Each chamber was filled with 1 ml of experimental or control test medium and incubated at 37°C. After 18-24 hr, the area of migration of cells in control and experimental media was determined by tracing the periphery of a magnified projection of the migrating cells on an Epoi LP-6 profile projector (Ehrenreich Photo Optical Industries, Inc., Garden City, N.Y.) and measuring it with a planimeter (LASICO, Los Angeles, Calif.). Percentage
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migration inhibition based on the average of four replicate samples was calculated as follows: % inhibition = 1 (
area in experimental supernatants x 100. area in control supernatants 1
In both migration inhibition assays, viability was measured by trypan blue exclusion and radiochromium release as previously described (3-6). In addition, both assaysshow reversibility; after continued incubation (48 to 72 hr) the migrationinhibited preparations regain the capacity for migration and become indistinguishable from controls. Tumor cell-endothelial cell binding assay. To study tumor cell binding to endothelial monolayers, tumor cell suspensions were obtained as described above. Endothelial monolayers were prepared from bovine pulmonary artery endothelial cells as described by Picciano et al. (13). Their identity as endothelial cells was confirmed by the detection of factor VIII antigen by indirect immunofluorescence (14). The tumor cells were labeled with “Cr by standard techniques (15). Tumor cells were resuspended at 5 X lo4 cells/ml in experimental or control supematant. Culture medium was aspirated from the wells of 12-well tissue culture plates (Falcon Plastics) and a l-ml suspension of tumor cells was added to each well. The plates were incubated at 37°C in a humidified 5% C02-95% air atmosphere for 60 min. Each culture plate contained samples in experimental and control media, thus providing an internal control for the consistency of the separation procedure. Upon completion of the incubation, nonadherent cells were removed by pipetting and the monolayers were washed four times with RPM1 1640 medium containing 2% FCS. After the last wash, the contents of each well, consisting of adherent labeled tumors and unlabeled endothelium, was solubilized by adding 1 ml of 0.5 N NaOH and the level of radioactivity was determined in a Beckman gamma counter. This procedure, which is a standard assayfor tumor binding ( 16), has good reproducibility, as seen in the very small standard errors in Table 2 (see below). RESULTS Initial screening for TMIF was performed using the agarose microdroplet assay which requires only small quantities of supernatant. Culture supematants from 36 hybridomas were tested. Cells from samples which showed marked TMIF activity were expanded to larger volumes. To prepare supernatants for further testing, l-2 X lo6 cells were placed in culture for 24 hr. The supernatants from these cultures were harvested and tested for both TMIF and MIF activity. In addition, supernatants were prepared from the myeloma fusion partner, BW5 147, and assayedfor inhibitory activity. The results in Table 1 show that supernatants prepared from BW5147 did not inhibit the migration of either tumor cells or murine PEC. Of the four other uncloned hybrids for which data is included, two (lB5 and 1ClO) could inhibit both tumor and macrophage migration. One hybrid (lB7) was negative for both activities. Supematants prepared from the fourth hybrid (3F4) could inhibit tumor cell but not macrophage migration. We prepared a number of clones from 3F4. The data derived from four of them are shown in Table 1. One cloned sample did not continue to release TMIF. Three
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SHORT COMMUNICATIONS TABLE I Effect of Hybridoma Supernatants on Tumor Cell and Macrophage Migration % Inhibition (mean f SE) Sample
p815”
PEC”
BW5 147 (fusion partner) IB5 IB7 IClO 3F4 2B6b 2C4’ 2C8’ 2C9’
0.2 ic 5.5 25.0 f 1.8 -5.3 f 4.7 20.2 f 4.6 38.2 f 3.9 2.9 zk 4.1 36.1 + 4.5 35.5 f 3.4 31.4 + 4.1
2.9 + 8.8 20.8 f 9.9 -9.5 f 10.2 24.8 f 6.9 15.5 + 7.0 nd’ 8.6 * 7.9 17.0 z!T 7.7 17.9 f 7.6
‘Values > 20% are considered to represent significant inhibition (6). Of the subclones, 2C4 has no activity against PEC, whereas 2C8 and 2C9 have marginal activity. All three, however, are active against P815. bCloned from 3F4. ’ nd = not done.
other clones behaved in the same manner as the hybrid from which they were derived. Supernatants from 2C4, 2C8, and 2C9 continued to releaseTMIF but only 2C4 did not affect macrophage migration. Clones 2C8 and 2C9 appeared to release marginal quantities of macrophage MIF. This pattern has continued. In all migration inhibition assays,viability was AO% with no difference between experimental and control preparations. Migration-inhibited preparations showed reversibility of effect; continued incubation for an additional 48-72 hr led to recovery of the capacity for migration. We next attempted to determine whether these supernatants exerted other noncytotoxic effectson tumor cell behavior. We have recently found that lymphokineTABLE 2 Effect of Hybridoma Supernatants on Tumor Cell Adherence to Endothelial Monolayers % Adherence (mean + SE; n = 4)b Supernatant Complete medium Unstimulated normal lymphocytes BW5 147 1B7 2B6 2C8
TMIF”
Expt I
Expt 2
Expt 3
72.3 f 0.9
75.1 * 4.0
16.4 f 4.4
74.8 f 50.6 f 39.4 + 47.0 + 54.0 f
72.9 5 2.8 54.7 f 3.6 (27.2) nd nd 46.3 + 4.2 (38.3)
69.3 f 58.5 + 52.3 k 63.9 f
1.6 1.2 (30.0) 1.0 (45.5) 3.7 (35.0) 2.0 (25.3)
nd 2.1 (9.3) 2.7 (23.4) 3.8 (31.5) 1.5 (16.4)
’ For comparison, the presence or absence of TMIF is indicated for each supematant. b% Adherence determined by radiolabel assaydescribed in the text. Figures in parenthesesrepresent % inhibition of adherence relative to medium control.
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containing supernatants derived from a lymphoblastoid long-term cell line as well as supernatants prepared from Con A-stimulated murine spleen cells known to contain TMIF were capable of suppressing the attachment of tumor cells to endothelial monolayers (17). It was, therefore, of interest to see if supematants prepared from hybridoma sources of which one member of the pair was a Con Aactivated spleen cell could behave in a similar fashion. The results in Table 2 indicate that hybridoma-derived supernatants could decreasetumor cell adherence. However, this did not correlate with the presence of TMIF. Furthermore, the fusion partner, BW5147, was also capable of modifying attachment of tumor cells to endothelial monolayers. DISCUSSION The ability to use hybridoma technology to produce clones of cells derived from one cell is of great potential in determining whether effector lymphokine molecules are responsible for more than one activity. A number of lines have been prepared which appear to secrete either one or only a very limited number of molecules (9-l 1, 18, 19). We have fused Con A-activated murine spleen cells, which normally produce migration inhibitory factors, with BW5 147 to produce a hybridoma which secretes a product capable of modifying the migration of neoplastic but not inflammatory cells as well as a number of other hybridomas that secrete products capable of inhibiting both. The ability to separatethese two lymphokine effectsis more than just an academic exercise. First, the question of whether one cell is limited to the production of one lymphokine has not yet been answered, since it is known that some lymphokines or cytokines can have multiple activities (20-23). Second, while it would be desirable to use migration inhibitory factors to prevent neoplastic cells from spreading throughout the organism, their use would be limited if inflammatory cells were affected as well. Our results indicate that it is possible to prepare supematants with TMIF activity devoid of MIF activity. Whether this indicates that these are two distinct molecular speciesis not clear because we have not been able to obtain lines of cells with MIF but not TMIF activity. Such a reciprocal finding would unequivocally exclude differences in assay sensitivity as a cause of the observed results. However, we have already found such reciprocal dissociations in our physicochemical characterization studies (3-5). Thus, incubation with fucose and rhamnose abolishes MIF but not TMIF activity, while conversely, diisopropyl fluorophosphate (DFP) inhibits TMIF but not MIF. Under appropriate conditions of culture, some murine strains produce TMIF in the absence of MIF, whereas guinea pig cells produce MIF but not TMIF. More evidence that the results reported here are not due to differences in assay sensitivity is provided by the observation that both activities are of approximately the same magnitude in activated normal lymphocyte cultures. Also, we never saw MIF activity reappear in the clones derived from 3F4, which would be expected if lack of MIF activity were due simply to different amounts of factors secreted. Finally, the previously reported size differences between TMIF and MIF provide further evidence for their nonidentity. However, it should be noted that absolute proof that TMIF can be produced in the absenceof MIF must await the development
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547
of a sensitive radioimmunoassay for both. Moreover, it is also possible that TMIF could be a subunit of the larger MIF molecule; i.e., the entire molecule is not necessaryfor an effect on tumor cells. There is evidence (24) that MIF is composed of two subunits, one of which is required for attachment to the target cell while the other exerts the observed biological effect. The latter is found in the same molecular weight range as TMIF and it is tempting to speculate that they might be identical or related. We have recently extended our studies of noncytotoxic lymphokine effects on tumor cells and have found a factor which causesdecreasedbinding of tumor cells to endothelial cell monolayers (17). This factor is found in supernatants known to contain TMIF activity, and in preliminary studies, appears to exert its effect on the tumor cells themselves. We found the activity in all the hybridoma supernatants that we tested, including supernatants prepared from the BW5 147 lymphoma line used for fusion. In contrast, we, and others, have never found migration inhibitory activities present in supematants prepared from this cell line (Table 1; Ref. (9)). Thus, it is possible the migration inhibitory activity and the binding inhibitory activities are distinct. Further studies will be required to confirm whether these activities are, in fact, due to different factors. It is clear that TMIF production is not merely a property of all growing cells, since the fusion partner, BW5147, was itself unable to produce TMIF. Also, we have previously reported that antigen-activated (3) or mitogen-activated (4) lymphocytes, but not unstimulated ones, produce TMIF. The situation for tumor-cell binding is less clear since, as indicated above, BW5 147 does produce a factor that inhibits tumor cell binding to endothelium. However, as seenin Table 2, nonactivated normal lymphocytes do not. Although antigen-activated murine lymphocytes also produce this binding inhibitory activity, antigen-activated guinea pig lymphocytes do not (unpublished observation), providing further evidence that this activity is not a nonspecific effect of cell culture supematants. Our present results demonstrate that T-cell hybridomas can be generated which constitutively produce factors which affect the behavior of neoplasms but are not cytotoxic for these cells. These cell lines represent continuous sources of mediators which have the potential for being relatively free of contamination by mediators with similar biologic behavior for other cell types. They therefore provide a convenient source of mediator for both purification and studies of potential therapeutic effect in viva REFERENCES 1. Ristow, S., and McKhann, C., In “Mechanisms of Tumor Immunity” (I. Green, S. Cohen, and R. T. McCluskey, Eds.). Wiley, New York, 1977. 2. Herberman, R. B., and Holden, H. T., A& Cancer Rex 27, 305, 1978. 3. Cohen, M. C., Zeschke, R., Bigazzi, I’. E., Yoshida, T., and Cohen, S., J. Immunol. 114, 1641, 1975. 4. Cohen, M. C., Goss, A., Yoshida, T., and Cohen, S., J. Immunol. 121, 840, 1978. 5. Cohen, M. C., Cancer Rex 42, 2135, 1982. 6. Adelman, N., Hasson, M., Masih, N., and Cohen, M. C., J. Immunol. Methods 34, 235, 1980. 7. Donskoy, M., Forouhar, F., and Cohen, M. C., Cancer Rex 44, 3870, 1984. 8. Cohen, M. C., Forouhar, F., Donskoy, M., and Cohen, S., Clin. Immunol. Immunopathol. 34, 94, 1985. 9. Jones, C. M., Braatz, J. A., and Herberman, R. B., Nature (London) 291, 502, 1981.
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10. Schreiber, R. D., Altman, A., and Katz, D. H., J. Exp. Med. 156, 677, 1982. 11. Erickson, K. L., Cicurel, L., Gruys, E., and Fidler, I. J., CeN. Immunol. 72, 195, 1982. 12. Beezley, B., and Ruddle, N. H., J. Immunol. Methods 52, 269, 1982. 13. Picciano, P. T., Johnson, B., Walenga, R. W., Donovan, M., Borman, B. J., Douglas, W. H. J., and Kreutzer, D. L., Exp. Cell. Rex 151, 134, 1984. 14. Ryan, U. S., Clements, E., Habliston, D., and Ryan, J. W., Tissue Cell 10, 535, 1978. 15. Munger, W., and Lindquist, R., In “Investigation of Cell-Mediated Immunity” (T. Yoshida, Ed.). Churchill Livingstone, Edinburgh, in press. 16. Kramer, R. H., and Nicolson, G. L. Proc. Natl. Acad. Sci. USA 76, 5704, 1979. 17. Cohen, M. C., Mecley, M., Antonia, S. J., and Picciano, P. T., submitted for publication. 18. Higuchi, M., Asada, M., Kobayashi, Y., and Osawa, T., Cell. Immunol. 78, 257, 1983. 19. Kaltmann, B., Gemsa, D., Hultner, L., Kees, U., Marcucci, F., and Krammer, P. H., In “Interleukins, Lymphokines and Cytokines” (J. J. Oppenheim and S. Cohen, Eds.). Academic Press,New York, 1983. 20. Hefeneider, S. H., Conlon, P. J., Henney, C. S., and Gillis, S., J. Immunol. 130, 222, 1983. 21. Atkins, E., J. Infect. Dis. 149, 339, 1984. 22. Steeg, P. S., Moore, R. N., Johnson, H. M., and Oppenheim, J. J., J. Exp. Med. 156, 1780, 1982. 23. Wong, G. H. W., Clark-Lewis, I., McKimm-Breschkin, J. L., Harris, A. W., and Schrader, J. W., J. Immunol. 131, 788, 1983. 24. Possanza,G., Cohen, M. C., Yoshida, T., and Cohen, S., Science (Washington, D.C.) 205, 300, 1979.