injection of the treated cells. As little as 1 antiviral unit of recombinant IFN-y per ml induced B16 cells to form 3-40 pulmonary metastases in each injected mouse, ...
Proc. Nati. Acad. Sci. USA Vol. 84, pp. 3405-3409, May 1987 Medical Sciences
Interferon y induces lung colonization by intravenously inoculated B16 melanoma cells in parallel with enhanced expression of class I major histocompatibility complex antigens (experimental metastasis/interferons/H-2 antigen/natural killer cells)
K. TANIGUCHI*t, M. PETERSSONt, P. HOGLUND*, R. KIESSLINGt, G. KLEIN*, AND K. KARRE*§ Departments of *Tumor Biology and tImmunology, Karolinska Institute, Box 60 400, S-104 01, Stockholm, Sweden
Contributed by G. Klein, December 1, 1986
Treatment of H-2-deficient nonmetastatic ABSTRACT B16 melanoma cells with physiological doses of interferon y (IFN-y) reduced cellular growth in vitro but induced a shift to the lung-colonizing phenotype as assessed after intravenous injection of the treated cells. As little as 1 antiviral unit of recombinant IFN-y per ml induced B16 cells to form 3-40 pulmonary metastases in each injected mouse, whereas a 1000-fold higher concentration of IFN-p was required to see similar effects. IFN-y may induce cell-surface molecules that contribute to the metastatic ability of the tumor cells. The efficient enhancement of metastatic ability after IFN-y treatment of the B16 cells was paralleled by an increased H-2 antigen expression and decreased sensitivity to natural killer cells. The experiments support the idea that metastasis may not depend exclusively on stable genetic changes or heterogeneity within a tumor population but may be also influenced through the modulation of the phenotype by physiological or pharmacological agents. The results are also discussed with regard to the role of different effector cells in tumor cell clearance and in relation to lymphokine-based strategies for therapy.
Changes in the phenotypic profile of metastatic, compared to primary, tumor cells (1-6) have been taken to suggest that tumor spread may occur through the selective outgrowth of rare but stable variant cells that can survive and grow in new host compartments. Membrane changes in such cells may influence their ability to penetrate the basal membrane, to invade adjacent tissues, to dissociate and circulate in the bloodstream, and to colonize distant organs (1-2). However, variations in surface properties within a tumor clone may also arise by reversible modulation that can be influenced by physiological or pharmacological agents in addition to stable genetic or epigenetic changes. This possibility has been explored only to a limited extent in studies of the different steps in tumor spread. Interferons (IFNs) are particularly notable among known cell-surface modulators, due to their potency and frequent use in experimental (7) or clinical (8) therapeutic attempts. During our studies on experimental metastasis we observed that a cultured subline of the B16 melanoma had a markedly reduced H-2 expression and was unable to colonize the lungs after intravenous inoculation into syngeneic mice (9). Treatment of the melanoma cells with an IFN-13-containing preparation (103-104 units/ml) before inoculation was followed by the appearance of a few metastatic colonies in each recipient mouse, as also found earlier in the Lewis lung carcinoma system (4). Different IFNs are known to vary in their ability to modulate cellular responses (10). IFN-y is of particular interest, since it is a potent inducer of major histocompatibility complex (MHC) antigen expression (10) and can modThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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ulate various immune functions (11, 12). It has been suggested that the efficient induction of MHC class I antigens by IFN-y may lead to an antineoplastic effect, by facilitating immune rejection, mediated by MHC-restricted T cells (13). This potential may be counteracted by the increased natural killer (NK)-mediated elimination of cells with reduced MHC expression, as observed in some systems (14, 15). In contrast to the concept of MHC-restricted T-cell recognition that predicts enhanced immune control and reduced malignancy of IFN-y-treated tumor cells, the preferential NK lysis of certain tumor cells with a reduced MHC expression (9, 14-18) predicts the opposite. We have tested the validity of these predictions in relation to the lung-colonizing ability of intravenously inoculated B16 melanoma cells.
MATERIALS AND METHODS Mice. CBA and C57BL/6 were bred in the animal colony of our institute. Tumor Cell Cultures and Treatment with IFN. B16 is a melanoma line originally derived from the C57BL strain (H-2b). MBA is a fibrosarcoma line induced by methylcholanthrene in the CBA strain (H-2k). Semiconfluent cultures of B16 or MBA cells (maintained in RPMI medium supplemented with 10% fetal calf serum, penicillin, and streptomycin = complete medium) were grown in Falcon tissue culture flasks (25 cm2) in the presence of different doses of IFN-a/IFN-,l [preparation from Ehrlich ascites cells, >107 international reference units (IRU)/mg of protein; Enzo Biochemicals, New York], IFN-/3 (preparation from murine L cells, 3.6 x 107 IRU/mg of protein; Stratech Scientific, London, no. 83001), or IFN-y (recombinant Escherichia coli-derived, from Genentech, South San Francisco, kindly provided by G. R. Adolf, Ernst-Boehringer Institut, Vienna). Specific activity of this IFN, measured against a mouse IFN-a/IFN-,l standard, was 7.2 x 106 units/mg of protein, as estimated by Genentec. The antiviral activity of the IFN preparations were confirmed in our laboratory against L-929 cells plated to confluency in 96-well microtiter plates and exposed to 1:2 dilutions of IFN-containing preparations. After incubation at 37°C overnight, the monolayers were challenged with 50 plaque-forming units of vesicular stomatitis virus and incubated 48 hr. Cells were scored for reduction in cytopathic effect and IFN titers were calculated against a National Institutes of Health a/,B reference IFN standard. Antiserum to IFN-p8 (sheep antiserum to mouse L-cell IFN; National Institutes of Health reference reagent) or IFN-y (rabbit anti-mouse, kindly provided by H. Johnson, Galveston, TX) Abbreviations: MHC, major histocompatibility complex; IFN, interferon; NK, natural killer; IRU, international reference units; FACS, fluorescence-activated cell sorter. tPresent address: Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. §To whom reprint requests should be addressed.
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was added in control experiments. After 24 hr, cells were washed in phosphate-buffered saline (PBS), gently trypsinized (2 min, 0.10% trypsin/EDTA solution), washed twice, and then used for further experiments described below. Experimental Metastasis. Cells (5 104 or 105) were inoculated into the tail vein of C57BL mice. The mice were sacrificed after 21-28 days, and a complete autopsy was performed. The colonies on the surfaces of the pulmonary lobes were counted under a dissection microscope. Fluorescence-Activated Cell Sorter (FACS) Analysis. Cells (5 x 105) were incubated with 100 Al of hybridoma supernatant [20-8-4S (H-2KbDb specific), 28-13-3S (Kb specific), or 28-14-8S (Db specific), all described in ref. 19 and obtained from the American Type Culture Collection] on ice for 45 min. The cells were washed and resuspended in fluoresceinconjugated rabbit anti-mouse immunoglobulin (Dakopatts, Stockholm), 1:10 dilution in RPMI medium, for an additional 45 min on ice. After three washes the cells were analyzed on a FACS IV, using a laser light output of 200-mV photomultiplier at 450 V and fluorescence gain at x4. In Vitro Growth. The cells were plated in complete medium in Falcon flat-bottom 96-well microtiter plates at a concentration of 2.5 x 104 ml (5 x 103 cells per well) and incubated in 370C in 5% CO2 in air. After 4 days, the wells were washed in PBS, trypsinized, and counted in trypan blue solution. Means standard deviation from triplicate wells were calculated after normalization of the data to the number of cells in control cultures without IFN treatment (=1.0). Sensitivity to NK Cells. The NK assay was performed with spleen cells from poly(I/C)- or tilorone-treated C57BL mice, as described (20). The tests were harvested after 4 hr. X
±
RESULTS IFN-a/IFN-/3 preparation derived from Ehrlich ascites cells and L-cell-derived IFN-/3 increased the lung-colonizing ability of B16 and MBA tumor cells (Table 1). Recombinant IFN-y was much more potent, however, since treatment of B16 cells with as little as 1 antiviral unit/ml increased the lung-colonizing ability of B16 cells. Ten units/ml led to a further increase (Fig. 1). Rabbit anti-IFN-y antiserum treatment totally abrogated the ability of the IFN preparation to enhance metastasis (Fig. 1). IFN-13 had no effect on the number of colonies at dose levels between 1 and 102 units, and treatment with 103 units/ml only gave a marginal increase in number of colonies (Fig. 1). IFN-y was thus at least 1000 times more efficient than IFN-1. In addition, 4 of 12 mice receiving B16 cells treated with 10 units of IFN-y per ml developed multiple (7-22) liver colonies (not shown). No other extrapulmonary metastases were observed.
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IFN-y was also superior to IFN-3 in its ability to enhance the expression of MHC class I antigens as measured by FACS analysis with monoclonal antibodies that react with common epitopes on the Kb and Db products (Figs. 1 and 2). This was due to an increase in H-2Kb as well as H-2Db determined by antibody 28-13-3S and antibody 28-14-8S (19), respectively (not shown). IFN-f3 enhanced class I antigen expression only at a dose of 103 units/ml (Fig. 1), confirming earlier reports on the effect of IFN-y vs. IFN-P on hematopoietic lines (10, 21). I-Ab antibody staining barely exceeded the background level and remained unchanged after IFN-y or IFN-,3 treatment (not shown). Lung colonization increased after exposure to low doses of IFN-y in spite of the fact that growth of the treated cells was inhibited in vitro (Fig. 1). IFN-p inhibited in vitro growth only at a dose of 103 units/ml. This was the only dose that induced lung colonization in vitro (Fig. 1). IFN treatment of tumor cells can induce resistance to NK cells ("protection"; refs. 20-23). IFN-/3 protected the melanoma cultures to 30-50%o but only at a dose level of 103 units/ml (Fig. 1). One unit of IFN-'y was already protective to >50%6, thus confirming previous findings on human hematopoietic lines (21). Treatment with 10 units/ml gave nearly complete protection.
DISCUSSION Our results show that a host mechanism favored the survival and/or lung colonization of IFN-y-treated, intravenously inoculated melanoma cells, in spite of the fact that the treatment inhibited their growth in vitro and increased their surface H-2 expression. This is at variance with the notion that a high expression of MHC class I antigens facilitates the recognition and elimination of tumor cells by MHC-restricted killer T cells (24, 25). The role of specifically sensitized T cells in tumor cell rejection is variable, however, depending on the presence of specific antigens on the tumor cell surface, which are competent to generate a strong adaptive response. Such antigens are only expressed on a minority of tumors (24, 26). Metastatic spread of certain transplantable tumors can be inhibited by NK cells (27). NK sensitivity of different tumor lines varies considerably, but it does not appear to be dependent on the expression of specific antigens. Treatment of tumor cells with IFN can induce resistance to NK lysis in vitro and NK-mediated elimination in vivo (20-23). Our data show that 10 units of IFN-y per ml increased the H-2 expression and made them completely resistant to NK lysis, in contrast to untreated B16 cells that did not express H-2 antigens and were NK sensitive (Fig. 1).
Table 1. Effect of IFN-/3 or IFN-a/IFN-,( on lung colonization Mice with pulmonary Tumor Exp. Treatment Units/ml colonies, no. Colonies,* no. 1 B16 1/9 4, 0, 0, 0, 0, 0, 0, 0, 0 6/7 8, 3, 2, 2, 1, 0 IFN-a/IFN-P 4 x 103 2 B16 0/4 0,0,0,0 IFN-a/IFN-13 4 x 103 4/4 3, 2,1, 1 3 B16 0/4 0, 0, 0, 0 1 x 103 4/4 16, 6, 4, 2 IFN-,B 4 MBA 1/6 1, 0, 0, 0, 0, 0 4x 103 4/6 6,4,3,2,0,0 IFN-,B 5 MBA 0/3 0, 0, 0 4 x 103 IFN-13 3/4 70, 5, 4, 0 6 MBA 3/5 12, 3, 2, 0, 0 IFN-a/IFN-(3 4 x 103 3/3 >200, 65, 55 *Number of pulmonary colonies in syngeneic mice 21 or 28 days after intravenous inoculation of 5 x 104 tissue culture-derived cells (105 cells in experiment 3), with or without treatment of the tumor cells with IFN.
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anti-IFN-,8 anti-IFN-y Antiviral units (IRU)/ml FIG. 1. Effects of IFN-8 and IFN-y on B16 melanoma cells. Cells were cultured in the presence of different doses of IFN for 36 hr, trypsinized, and washed prior to all assays. NT, not tested. Pulmonary colonies: diagram shows number of colonies in intravenously inoculated individual mice in two independent experiments (x, *). FACS analysis: columns represent the median channel of fluorescence intensity in an indirect assay with an H-2 KbDb antibody as the primary reagent; background median values with the secondary reagent (fluorescein isothiocyanate-conjugated rabbit anti-mouse Ig) varied between 15 and 20. NK sensitivity: % specific lysis at effector/target ratios of 40:1 (X) and 20:1 (e), with spleen cells from mice treated with an IFN inducer. In vitro growth: columns represent the cell number recovered 4 days after termination of the IFN treatment and replating, relative to untreated cells.
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Fluorescence intensity (linear) FIG. 2. FACS profiles showing H-2KbDb expression after IFN-y IFN-y, 1 IRU. (C) IFN-y, 10 IRU. (D) IFN-y, 10 IRU, and anti-IFN-y. Experimental procedure and reagents as in Fig. 1. treatment of B16 cells. (A) Control. (B)
There was a striking similarity between the dose-response for NK protection, increase in H-2 expression, and enhanced lung colonization after IFN-y or IFN-/3 treatment, and all effects were neutralized by the appropriate anti-IFN antiserum (Fig. 1). It has been shown in several experimental systems that the malignant phenotype can be associated with enhanced or de novo expression of certain H-2 antigens (3-6). We have found that lymphoma variants selected for loss of H-2 expression differed from the original high H-2 expressor line in showing an increased sensitivity to natural killing and a reduced ability to colonize the lungs and the liver after intravenous injection (14, 16). In contrast to the H-2positive parental lines, they could not be protected from NK lysis by IFN-P or IFN-y, although they did respond to other IFN-mediated effects (18). It is therefore conceivable that the increased H-2 expression, NK resistance, and switch to the lung-colonizing phenotype in IFN-y-treated B16 cells represent causally related, rather than coincidental, events. IFNinduced H-2 antigen expression on the tumor could influence their interactions with other cells and particularly facilitate their escape from NK cells. This is supported by our earlier findings on other tumors (14-18). The situation may be entirely different for systems in which T cells dominate the tumor-host interaction-e.g., in tumors that express strong specific antigens. It is noteworthy in this context that a gain of MHC class I expression was associated with the loss of malignancy in B16 melanoma variants that had become immunogenic for the T-cell system after mutagenization (28). The question of whether the lung colonization by our IFN-y-treated B16 cells can be entirely accounted for by their increased H-2 expression remains to be analyzed. It is nevertheless clear that differences in metastatic ability do not necessarily reflect stable genetic or epigenetic heterogeneity within a tumor cell population but may be also generated by modulations through physiological factors. It has been suggested that local levels of IFN-y may be important for immunoregulation (29). With due consideration of the fact that our results have been obtained in a limited experimental system, they raise the possibility that IFN-y may contribute to organ selectivity of metastasis (1). Local immune responses mediated by IFN-y-producing T cells might thus enhance, rather than inhibit, tumor spread under certain conditions. It
curves
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is noteworthy that in human melanoma, metastasis and poor prognosis are associated with high MHC class II expression in the primary tumor (30). The presence of such elevated HLA-DR levels is itself associated with mononuclear cell infiltrates located in close contact with the tumor cells (31, 32) and with IFN-y in these infiltrates (33). An evaluation of the different IFN preparations, based on the tumor-inhibitory effect in vitro, would have predicted the opposite of what was actually observed in vivo. Our results emphasize that proliferative and metastatic properties are not necessarily linked. Metastasis is a complex process, involving sequential interactions between tumor cells and the host environment. The present study focused on how the IFN-induced B16 melanoma phenotype behaves in the events following penetration into blood vessels. It is conceivable that certain tumor cell properties that favor the spreading process at a particular stage-e.g., in the vascular bed-may inhibit growth after organ colonization has been accomplished. The temporary induction of such properties may thus promote metastasis more effectively than the selection of a corresponding phenotype. Temporary modulation of the tumor cell phenotype by IFNs is of particular interest since they are frequently used in tumor therapy (7, 8). It cannot be excluded that even positive therapeutic net effects may reflect the compounded end result of IFN effects that promote tumor spread, counterbalanced by cytostatic and immunostimulatory effects. The use of lymphokines for the activation of cytotoxic cells in situ or in vitro for reinfusion represents a particularly interesting situation (34). It has been shown that T3-negative NK cells are the dominating cytotoxic effectors after peripheral blood lymphocyte cultivation in interleukin 2 (IL-2) (35, 36). If the present results apply also to human targets and NK cells, IL-2-based therapeutic strategy may be most efficient without simultaneous IFN-y (exogenous or IL-2 induced) influence on tumor cells that have reached the vascular bed. (Re)expression of H-2 antigens after DNA-mediated gene transfer was found to induce immunogenicity and inhibit metastasis formation in a murine fibrosarcoma (37). Reinduction of MHC antigens with IFN--y might be another way to facilitate host recognition and rejection of potentially metastatic cells (13). Although this can work with suitably immunogenic tumor cells, our findings question the generality of this approach. In preliminary studies, IFN--y had an analogous promoting effect on lung colonization by a methylcholanthrene-induced sarcoma of CBA origin but had no effect on a mammary adenocarcinoma of the same strain (P.H. and K.K., unpublished results). It will be important to define the parameters responsible for the contrasting effects of IFN-y on different tumors. We thank Dr. G. R. Adolf (Ernst-Boehringer Institut, Vienna) for kindly providing recombinant E. coli-derived IFN-y (Genentech), Ms. M. L. Solberg and Ms. M. Hagelin for technical assistance, Dr. H. G. Ljunggren for stimulating discussions, and Ms. A. Schmalholz for secretarial assistance. This investigation was supported by Public Health Service Grants 5 R04 CA25250-06 and 1 R01 CA26782-02, awarded by the National Cancer Institute, Department of Health and Human Services, Bethesda, MD, and by grants from the Swedish Cancer Society and the Swedish Society for Medicine. K.T. was supported by a fellowship from the International Union Against Cancer. 1. Nicolson, G. L. (1982) Biochim. Biophys. Acta 695, 113-173. 2. Poste, G. & Fidler, I. J. (1980) Nature (London) 283, 139-146. 3. De-Baetselier, P., Katzav, S., Gorelik, E., Feldman, M. & Segal, S. (1980) Nature (London) 288, 179-181. 4. Eisenbach, L., Segal, S. & Feldman, M. (1983) Int. J. Cancer 32, 113-120. 5. Katzav, S., De-Baetselier, P., Gorelik, E., Feldman, M. & Segal, S. (1981) Transplant. Proc. 13, 742-746. 6. Katzav, S., De-Baetselier, P., Tartakovsky, B., Feldman, M. & Segal, S. (1983) J. Natl. Cancer Inst. 71, 317-324.
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