In vitro effects on tumor cells of macrophages isolated from an early-passage chemically-induced...

Int. J . Cancer: 27, 221-228 (1981)

IN VITRO EFFECTS ON TUMOR CELLS OF MACROPHAGES

MURINE SARCOMA AND FROM ITS SPONTANEOUS METASTASES ISOLATED FROM AN EARLY-PASSAGE CHEMICALLY-INDUCED

Albert0 MANTOVANI Istituto di Ricerche Farmacologiche “Mario Negri”, Via Eritrea, 62-20157 Milan, Italy.

Macrophages were isolated by adherence from an early-passage chemically-induced tumor in C57BU6 mice and from its spontaneous lung metastases. Cytoly- tic activity was measured as release of [’HI-thymidine from prelabelled tumor cells and cytostasis in a post- labelling assay. Normal, unstimulated peritoneal macrophages exhibited low but significant non-specific cytotoxic activity at attacker to target (AT) ratios L 20: I, whereas stimulation of tumor-cell proliferative capacity was consistently observed at A T ratios 5 21. Mac- rophages from the peritoneal cavity of tumor-bearing mice, from the primary tumor or from pooled secondaries had baseline cytotoxic or growth-enhancing (de- pending on the A T ratio) potential similar to that of control peritoneal macrophages. In vitro exposure to endotoxin, lymphokine supernatants or partially purified fibroblast interferon augmented the tumoricidal activity of normal and tumor-associated macrophages; tumor-associated macrophages showed no evidence of enhanced responsiveness to these activating stimuli. Tumor cells from the primary neoplasm and its spontaneous metastases were equally susceptible to macrophage tumoricidal activity. It is inferred from in vitro data that the relatively low numbers of non-activated macrophages present within poorly immunogenic metastasizing tumors may have no direct effect or actu- ally stimulate tumor-cell proliferative capacity at the primary tumor site; conversely, disseminating tumor cells might encounter sufficient numbers of mononuclear phagocytes in the circulation and in lungs to per- mit the expression of macrophage cytotoxicity, at least during the early steps of metastasis formation.

Macrophages infiltrate murine and human neoplasms (Evans, 1967, 1972; Eccles and Alexander, 1974; Pross and Kerbel, 1976; Wood and Gollahon, 1977) and their relative concentration in tumors is reportedly related to tumor immunogenicity and to host immunological status (Eccles and Alexander, 1974; Pross and Kerbel, 1976; Mantovani, 1978). Macrophages can be cytotoxic in vitro on tumor cells either spontaneously (Keller, 1978; Mantovani et al., 1979a, 19780a; Tagliabue et al., 1979) or after exposure to chemical or biological stimuli (reviewed by Hibbs, 1976; Evans and Alexander, 1976). Augmen- tation of macrophage cytotoxicity in rodents has been defined as a multi-step event (Hibbs et al., 1977; Chapman and Hibbs, 1977; Russell et al., 1977; Ruco and Meltzer, 1978a, b) and macrophages from Moloney sarcoma virus-induced tumors have been reported to be in a stable non-cytolytic stage of activation enabling them to respond more readily to endotoxins (Russell et al., 1977). The suggested in vivo relevance of macrophage cytotoxicity either as a surveillance mechanism (Hibbs, 1976; Evans and Alexander, 1976; Mantovani et al., 1979a, 1980a) or in the control of established tumors and metastases (Birbeck and Carter, 1972; Eccles and Alexander,

1974; Pross and Kerbel, 1976; Russell and McIntosh, 1977; Mantovani, 1978) remains to be firmly established.

In spite of the possible in vivo significance of macrophages in the regulation of tumor growth and metastasis, little is known about monunuclear phagocytes associated with metastasizing neoplasms and with spontaneous secondaries. The present in- vestigation was designed to elucidate the functional status of macrophages isolated from an early-passage chemically-induced murine sarcoma and from its spontaneous lung metastases.

MATERIAL AND METHODS

Mice and tumors Male C57BW6 mice, 6 to 10 weeks old, were ob-

tained from Charles River Breeding Laboratories, Calco, Italy.

The MN/MCAl fibrosarcoma was induced in this laboratory in 1978 by S.C. injection of 1 mg 3-methyl- cholanthrene in a male C57BU6 mouse (Giavazzi et al., 1980). It was the only one of a series of similarly induced tumors to give spontaneous metastases. After i.m. inoculation of lo5 tumor cells a nodule was palpable on day 11. Median survival time of tumor-bearing mice was 39 days (range 27-49) and half of these had spontaneous lung secondaries at autopsy. The average number of metastases at death was 2.2 k 0.5 (sE). The tumor was weakly immunogenic as surgical excision of the growing neoplasm conferred protection against subsequent chal- lenge with lo4 cells. The MN/MCAl sarcoma was used at its 2nd-5th transplant generation after induction. The weight of the primary intramuscular tumor and the number and weight of lung secondaries were measured as previously described (Spreafico et al., 1975; Giavazzi et al., 1980; Mantovani et al., 1980b).

Peritoneal macrophages Unstimulated peritoneal exudate cells were ob-

tained by washing the peritoneal cavity with 4 ml basal medium Eagle (BME). Exudates from 10-20 mice were pooled and centrifuged at 400 g for 5 min. After resuspension at a concentration of 1-2 X lo6 cells/ml, 5 ml were seeded in 25 cm2 microexudate- coated tissue culture flasks (Costar, Cambridge, Mass.). Microexudate-coated plastic was obtained by growing cell lines to confluence as previously described (Ackerman and Douglas, 1978; Mantovani et al., 1979a); Tagliabue et al., 1979). After incubation for 45 min at 37”C, non-adherent cells were

Received: October 17, 1980.

222 MANTOVAN1

thoroughly washed off with jets of BME with the aid of a Pasteur pipette. Adherent cells were recovered from microexudate-coated plastic by incubation with 3 ml 1 mM ethylene diamine tetraacetic acid (EDTA) in phosphate-buffered saline (PBS) for 5- 10 min at 37°C. After vigorous resuspension with a Pasteur pipette, cells were washed with 20 ml BME and resuspended in RPMl 1640 medium with 10% fetal bovine serum (aseptically collected FBS, Mic- robiological Associates, Walkersville, Md., o r GIB- CO-Europe, Glasgow, Scotland) and 50 pgiml gen- tamicin (growth medium). More than 95% of the adherent cells belonged to the macrophage series, as assessed by morphology, avid uptake of neutral red, binding and phagocytosis of antibody-coated sheep erythrocytes.

Tumor-associated macrophages ( T A M ) TAM were obtained essentially as originally de-

scribed by Evans (1972, 1973). Solid tumors were obtained from five or six animals 15-25 days after inoculation of 104-105 sarcoma cells; they were minced with scissors and disaggregated by exposure for 30-45 min to 0.1 % trypsin or 0.3 % collagenase (Sigma Chemical Co., St. Louis, MO) in BME containing 10 pgiml DNase. Disaggregated cells were centrifuged and resuspended in BME at a concentration of 2-5X10' cellsiml. Five ml of the cell suspen- sion were seeded on microexudate-coated plastic as described above and incubated for 30 min at 37°C with agitation every 5-10 min. Non-adherent cells were thoroughly washed off with jets of BME with a Pasteur pipette. Adherent cells were recovered from conditioned plastic by exposure to EDTA as described above. More than 95 % of the adherent cells were macrophages as assessed by the above criteria. Contaminating cells were, in order of frequency, tumor cells, unidentified small mononuclear cells and polymorphs. In a few experiments, to obtain TAM of greater purity, adherent cells were cultured for 48h in serum-free BME, with a change of medium, before further washing with BME and de- tachment (Evans, 1972, 1973). Under these conditions, more thant 9 9 % of the adherent cells were macrophages using the above-mentioned criteria.

In two experiments, macrophages were isolated also from pooled spontaneous lung metastases. Mice were inoculated with lo4 tumor cells i.m. and killed 34 and 43 days after transplant. Metastases were dis- sected free of normal lung parenchyma and pooled from 40-60 animals. Nodules were washed extensively with BME, and, after squeezing with a 5-ml sy- ringe plug, disaggregated with trypsin as described above for the primary tumor.

Tumor target cells The simian virus 40-transformed mKSA TU5

(TU5, Kit et al., 1969), tumor line was used as standard target in cytotoxicity assays. The TU5 line was maintained in growth medium. In two experiments tumor cells from the primary MNiMCAl sarcoma o r from its lung metastases were used as targets at their 3rd or 4th in vitro passage in growth medium.

To label tumor cells for cytolysis assays, non-con- fluent cultures were maintained overnight at 37°C in

5 ml growth medium with 0.5 pCiiml [3H-methyl]- thymidine (6 Ci/mmole, The Radiochemical Centre, Amersham, Bucks., England) in 25 cm2 tissue culture flasks (Costar). Cells were detached by incubation for 5 min with 2 ml of 0.25% trypsin - 0.02% EDTA in PBS and washed twice with 50 ml BME before resuspension in growth medium.

Activation of macrophage cytotoxicity Stimuli used to augment macrophage cytotoxicity

included: endotoxin (Alexander and Evans, 1971); lymphokines (David, 1975; Piessens et a[., 1975; Fi- dler, 1975; Christie and Bomford, 1975; Ruco and Meltzer, 1977); interferon (IF) (Schultz el al., 1977; Jett et al., 1980; Mantovani et al., 1 9 8 0 ~ ) . Lym- phokine supernatants were the kind gift of Dr. Dia- na Boraschi (this Institute) and were prepared as previously described (Ruco and Meltzer, 1977; Tag- liabue et al., 1979). Briefly, spleen cells were obtained from mice killed 3-6 weeks after immuniza- tion with 2-3X10' cfu Mycobacterium bovis, strain BCG, intradermally. Five ml of the splenocyte sus- pension (5X 1O6/ml medium with 5 % FBS) were cultured for 4 h with 100 pg/ml purified protein deriva- tive of tuberculin (PPD, Connaught Medical Re- search Laboratories, Toronto, Canada, or Sclavo, Siena, Italy) or without PPD (control conditioned medium). The cells were washed and, after resuspension in fresh culture medium, were cultured for 48h at 37°C. Supernatants were harvested and stored at -20°C. Control conditioned media had no consistent effect on macrophage cytotoxicity.

Partially purified mouse fibroblast I F (lot 940065, l.2X107 units/mg protein) was purchased from Cal- biochem (La Jolla, C A . , USA). In a few experiments a crude mouse fibroblast I F preparation (Cat No. 407563, Calbiochem) was used. The two preparations had a comparable stimulatory effect on macrophage cytotoxicity. Endotoxin (W, Salmonella ty- phosa 0901) was obtained from Difco Laboratories, Detroit, MI., USA.

Cytolysis assay Cytolytic activity was measured as [ 3H]-thymidine

release from prelabelled target cells over a period of 48h as previously described (Mantovani et al., 1979a, 1980a,c). Briefly, tumor cells ( lo4) were incubated in 0.3 ml growth medium in 6.4-mm flat- bottomed tissue culture wells (3596, Costar, Cam- bridge, MA., USA) using an attacker to target cell (A:T) ratio of 10:1, unless otherwise specified. Tumor growth was checked daily under a n inverted microscope. Percentage isotope release was calcu- lated as 100 X AIB, where A is the isotope release in the supernatant and B is the total radioactivity re- leased by incubating target cells in 1 % sodium dodecyl sulfate in water. Specific release was deter- mined by subtracting spontaneous release in the ab- sence of effector cells which never exceeded 25 % of total incorporated radioactivity. Mean total incorporated radioactivity was 7,900, 4,200 and 3,100 cpm for TU5, primary and metastatic MN/MCAl sarcoma respectively. In most experiments performed, stimuli (lymphokine supernatant, I F and endotoxin) were present throughout the assay and did not affect

CYTOTOXICITY OF TUMOR MACROPHAGES 223

the viability or proliferative capacity of the targets (Mantovani et al., 1980a; Tagliabue et al., 1979).

Cytostasis assay Tumor-cell proliferative capacity was measured as

incorporation of [ 3H]-thymidine in a previously described post-labelling assay (Mantovani, 1978). Briefly, wells containing unlabelled tumor cells and macrophages were washed twice with 0.3 ml warm growth medium and cultured for 3 h with 0.2 ml growth medium containing 0.5 pCi [ 3H]-thymidine (1.9 Ci/mmole, Schwarz Mann, Orangeburg, NY). Controls consisting of macrophages alone were run in each experiment but isotope uptake by these cells was negligible (1500 cpm in wells with 2X105 macrophages) and was not taken into account.

Statistical analysis Results presented are mean k SD of 3-6 replicates

per experimental group. Significance was assessed by Duncan’s new multiple range test. Experiments were repeated at least three times, except for macrophages from metastases which were tested only twice.

10 20 30 40 5 0

D a y a f t e r t u m o r i m p l a n t a t i o n

FIGURE 1 - Growth, macrophage content and metastasis of the MNiMCAl sarcoma. Mice were given l o4 cells i.m. on day 0.

RESULTS

Between days 15 and 30 after inoculation of lo4 cells, the MN/MCAl sarcoma had a constant macrophage content of 30-40% of the disaggregated cells (12 experiments performed, Fig. 1). The percentage of TAM in the intramuscular lesion ap- peared to drop to 20% after day 30, but at these times the macrophage content of tumors was measured only twice. Pooled lung metastases had a macrophage content similar to that of the corresponding primary intramuscular lesions measured at the same time (Fig. 1).

The tumoricidal potential of TAM was then ex- amined. The TU5 line was routinely used as target for non-specific macrophage cytotoxicity as adherent effector cells against this tumor have previously been characterized (Mantovani et al., 1979a, 1980a; Tag-

liabue et al., 1979). Normal resident peritoneal macrophages, not deliberately exposed to stimuli, exhibited low but detectable levels of “spontaneous” cytotoxicity at A:T ratios greater than lO:l, specific isotope release values above background ranging from 7 to 21 % at a ratio of 20:l in the present series of experiments. A t A:T ratios S l O : l , normal macrophages had no baseline natural cytotoxicity. Occa- sionally, release values were lower in the presence of low numbers of macrophages (15:l) than with tumor cells alone (e .g . Fig. 2a) , but this was not a consistent finding and was usually observed when spontaneous release values of tumor cells alone ap- proached 20 % of total incorporated radioactivity. T A M and peritoneal macrophages from mice bearing an i.m. growing sarcoma had tumoricidal activity similar to that of control peritoneal macrophages (Fig. 2a).

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224

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dotoxin was present throughout the assay and, at these concentrations, did not affect spontaneous isotope release. The A:T ratio was 1 O : l .

When cytostasis was measured (Fig. 2b), inhibition of tumor-cell proliferative capacity by macrophages was observed at A:T ratios greater than 10:1, as expected o n the basis of the cytolysis data, whereas increased proliferation occurred in the presence of low numbers of effectors (1:l-2:l). Aug- mentation of tumor-cell proliferation was also observed in one experiment in which numbers of tumor cells in wells were evaluated by a spectrophotome- tric dye uptake test (Mantovani et al., 1980a,c). Macrophages from the peritoneal cavity of normal and tumor-bearing mice had similar growth-inhibit- ory (at A:T ratios2lO:l) and stimulatory (at A:T ratios 3 : l ) capacities.

Macrophages from MSV sarcomas have been reported to show enhanced responsiveness to endotoxin. Therefore, I studied the responsiveness of T A M from the poorly immunogenic, metastasizing MN/ MCAl sarcoma to lymphokines, IF and endotoxin (Figs. 3,4 and 5). These various stimuli augmented the tumoricidal capacity of both normal and tumor- associated macrophages. T A M showed no evidence of enhanced responsiveness to these activating agents, but occasionally (e.g., experiment in Fig. 4) boosting of cytotoxicity was somewhat lower with T A M than with control effector cells. In experiments presented in Figures 3-5, macrophages were exposed to stimuli throughout the cytolysis assay. Similar results were obtained when macrophages were activated by preincubation with graded doses of endotoxin (for 20h), IF (for 20h) or lymphokines (for 6h) (data not shown).

Experiments discussed so far were performed with T A M freshly isolated from tumors which contained an appreciable contamination of other cell types. In three experiments, TAM were maintained for 48 h in serum-free medium, a procedure which resulted in

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virtually pure preparations of mononuclear phagocytes (Evans, 1973; see “Material and Methods”). Results with these cells were similar to those discussed above, in that T A M had baseline cytotoxicity and responsiveness to stimuli similar to those of control effectors (results not shown).

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CYTOTOXICITY OF TUMOR MACROPHAGES 225

In two experiments the responsiveness of macrophages from lung secondaries was compared to those from the primary tumor tested concomitantly (Fig. 6). No difference was observed between the two sources of tumor-associated macrophages in their baseline cytotoxicity (A:T = 1O:l) and responsiveness to graded concentrations of endotoxin (Fig.

Because of the limited amount of tumor tissue obtained from secondaries, the low macrophage numbers available did not allow testing of different A:T ratios and different concentrations of other stimuli, but at an A:T ratio of 1O:l they were as responsive as TAM from the i.m. growing tumor to a single concentration of IF (300 unitdml) and lymphokines (1:81 final dilution) (results not presented).

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FIGURE 6 - Responsiveness to endotoxin of macrophages isolated from the i.m. growing tumor and its spontaneous lung metastases. Lung metastases were pooled from 44 animals 42 days after transplant of lo4 sarcoma cells i.m.

Results discussed so far were obtained using the TU5 tumor, unrelated to the MN/MCAl sarcoma, as target. This line was used as standard target in cytotoxicity assays as adherent effector cells against this tumor have previously been fairly extensively characterized and it appears relatively resistant to natural killer cell-mediated lysis (Mantovani et al., 1979a, 1980a; Tagliabue et al., 1979). Cells from i.m. growing MN/MCAl tumor were susceptible to macrophage cytotoxicity to an extent similar to T U 5 (compare Figs. 1-3 with Figs. 7 and 8). Moreover, in four experiments cells from the primary neoplasm or from spontaneous lung secondaries were tested after a limited equal number of in vitro passages three or four in different experiments) for their susceptibility

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tumor cells from the primary i.m. sarcoma and its spontaneous lung metastases. Unstimulated normal peritoneal macrophages were used as effectors. Results are mean of four experiments and expressed as percentage of specific lysis.

to macrophage tumoricidal capacity. As illustrated by the representative experiments shown in Figures 7 and 8, cells cultured from the i.m. growing sarco-

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226 MANTOVANI

ma or from its spontaneous lung secondaries were equally susceptible to baseline and endotoxin-activated peritoneal macrophage cytotoxicity.

DISCUSSION

The present study was designed to characterize the functional status of macrophages isolated from a poorly immunogenic, spontaneously metastasizing murine sarcoma of recent origin, and from its spontaneous lung metastases. Macrophages from the primary i.m. neoplasm and from lung secondaries did not significantly differ from normal peritoneal macrophages in terms of baseline spontaneous cytotoxicity (detectable at A:T ratios exceeding 10:1) and responsiveness to endotoxin, lymphokines and IF.

Several studies have focused on the functional status of mononuclear phagocytes in neoplasia and alterations of phagocytosis, migration and matura- tion of monocytes or macrophages have been described (Carr, 1977; Meltzer and Stevenson, 1978; Currie and Hedley, 1977; Nyholm and Currie, 1977). In several studies, macrophages from the lym- phoid organs or primary tumors of rodents bearing chemically or virally-induced neoplasms have been reported to show increased cytostatic andlor cytolytic activity against tumor cells in vitro (Kirchner et al . , 1975; Mantovani et al., 1977; Puccetti and Hol- den, 1979; Taniyama and Holden, 1979; Russell and McIntosh, 1977). On the other hand, no evidence of enhanced cytotoxicity was obtained with TAM from a long-term-passaged, weakly immunogenic, metastasizing murine tumor (Mantovani, 1978) and from human ascitic (Mantovani et al. , 19796, 19806) and solid (Mantovani A., unpublished data) ovarian tumors. Thus, the lack of macrophage activation in the present study may simply be a reflection of the biological properties of the tumor under study, i.e. poor immunogenicity, metastasizing capacity, and recent origin. The enhanced cytotoxicity andlor responsiveness to activating stimuli of macrophages in strongly immunogenic non-metastasizing tumors such as Moloney sarcoma virus-induced sarcomas may be a reflection of an ongoing immune response against the neoplasm (Taniyama and Holden, 1979). An additional factor, unrelated to neoplasia, which might alter macrophage functional status is repre- sented by the lactate dehydrogenase virus (LDV), a common contaminant of long-term transplanted tumors and viral preparations. LDV has been shown to alter macrophage cytotoxicity and chemotactic responsiveness (Evans and Alexander, 1976; Steven- son et al. , 1980).

Low concentrations of normal and tumor-associated macrophages (A:T ratios < 5:l) augmented tumor cells’ proliferative capacity. A feeding effect of rodent peritoneal macrophages on tumor cell proliferation has previously been observed (Namba and Hanaoka, 1972; Hewlett et al. , 1977; Krahenbuhl et al., 1976; Olivotto and Bomford, 1974; Buick et al. , 1980) particularly when low A:T ratios were employed (Keller, 1973, 1976; Mantovani et al., 1979a,b) or when tumors were maintained in poor culture conditions (Nathan and Terry, 1975; Evans, 1976). In vitro stimulation of tumor-cell proliferative

capacity was also reported with TAM from human ovarian tumors (Mantovani et al., 19796, 19806) and from a metastasizing variant of a murine sarcoma (Mantovani, 1978). However, at variance with the latter study in which TAM were better stimulators than normal peritoneal macrophages, the present in- vestigation showed comparable augmentations of tumor cell proliferation with the various effectors employed.

It has been suggested that selection of variant cells might play a role in metastasis formation (Fidler, 1978). Thus, tumor cells seeding and growing in lungs, a macrophage-rich anatomical site, might have been selected for resistance to macrophage cytotoxicity andlor for capacity to grow with minimal accumulation of macrophages. Pooled metastatic nodules had a macrophage content similar to that of the i.m. growing tumors tested concomitantly. Moreover, cells from pooled metastases showed no increase in resistance to the cytotoxicity of normal or activated macrophages. Thus it appears that, within the time course of spontaneous metastasis in this murine model, no appreciable selection for altered interaction with macrophages occurred.

The in vivo relevance of the in vitro effects on tumor cells of TAM remains to be elucidated. The murine sarcoma used in the present study has a macrophage content of 30-40%. This would result in A:T ratios in vivo at which TAM have little or no cytotoxicity in vitro. Hence, one might infer from in vitro data that TAM either have no direct influence on primary tumor growth in vivo or result in a net stimulatory effect. An in vivo stimulation of tumor growth by TAM has been suggested by Evans (1977a,b) on the basis of the effect of depletion of TAM with azathioprine or irradiation. Moreover, administration of the macrophage toxins silica and carrageenan resulted in decreased growth of some murine tumors (Sadler et al., 1977; Mantovani et al., 1980b). Thus the hypothesis that the relatively low number of macrophages present within poorly immunogenic metastasizing tumors, with cytotoxic potential similar to that of normal effectors, may not restrain tumor growth but even act as a mechanism of stimulation of cancer cell proliferation, merits serious consideration. Conversely, disseminating tumor cells might encounter in the circulation or at macrophage-rich anatomical sites (e .g . lungs) sufficient numbers of mononuclear phagocytes to result in consistent killing of metastasizing cells and in re- straint of metastases, at least during the early phases of metastasis formation. The divergent effects of macrophage toxic agents on growth of some i.m. or S.C. tumors and on their spontaneous metastases (Mantovani et al., 19806, Sadler et al., 1977) would appear to support this possibility.

ACKNOWLEDGEMENTS

This work was supported by grant ROI-CA 12764 from the Division of Cancer Treatment, National Cancer Institute, USA. I wish to thank Dr. R. Giavazzi, Dr. G. Alessandri and Mrs. N. Polentarut- ti for providing tumor-bearing mice and tissue culture lines.

CYTOTOXICITY OF TUMOR MACROPHAGES

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