Int. J . Cancer: 22, 741-746 (1978)

EFFECTS ON IN VITRO TUMOR GROWTH OF MURINE MACROPHAGES ISOLATED FROM SARCOMA LINES DIFFERING IN IMMUNOGENICITY AND METASTASIZING CAPACITY

Albert0 MANTOVANI Istituto di Ricerclze Farmacologiche ‘‘ Mario Negri ”, Via Eritrea, 62-20157 Milan, Italy

Macrophages were isolated from the strongly immunogenic, non-metastasizing FS6 sarcoma trans- planted in syngeneic C57BL/6 mice and from a weakly immunogenic, spontaneously metastasizing line (mFS6) of the same tumor. Macrophages isolated from the FS6 sarcoma non-specifically inhibited growth and DNA synthesis of tumor cells in vitro. After culture FS6 macrophages rapidly lost their cytotoxic activity and became capable of non- specifically stimulating tumor growth in vitro. Macrophages obtained from the mFS6 tumor non- specifically enhanced the proliferative activity of tumor target cells and this capacity was maintained when the macrophages were cultured.

Macrophages exposed in vitro or in vivo to chemi- cal or biological stimuli can be non-specifically cytotoxic in vitro on tumor target cells (reviewed by Evans and Alexander, 1976). Macrophage cyto- toxicity (lysis or stasis) appears to be preferentially expressed on tumor cells or virus-infected normal cells (Hibbs, 1974; Cleveland et al., 1974; Goldman and Hogg, 1978). Although the in vivo significance of macrophage cytotoxic activity as a defence mechanism against neoplasia is far from clear, there is evidence supporting the hypothesis that macrophages play a role in the regulation of tumor growth and metastasis (Birbeck and Carter, 1972; Eccles and Alexander, 1974; Pross and Kerbel, 1976, Russell and McIntosh, 1977; Wood and Gillespie, 1975 ; Zarling and Tevethia, 1973).

Macrophages have been shown to infiltrate rodent and human tumors and their concentration in experimental systems appears to be related to tumor immunogenicity and metastasizing capacity (Eccles and Alexander, 1974). Tumor-associated macrophages can be cytotoxic in vitro on tumor cells in a specific or usually non-specific manner (Evans, 1976; Holden et al., 1976; Russell and McIntosh, 1977) but the relationship between this macrophage cytotoxicity and the in vivo properties of tumors is still obscure. Evans, investigating the in vitro cytotoxic activity of macrophages from different rat chemically-induced sarcomas main- tained in vitro for 24 or 48 h, found no correlation between cytotoxicity and metastasizing capacity (Evans, 1976). On the other hand, in mice bearing Moloney sarcoma virus (MSV) induced sarcomas, macrophages isolated from regressing tumors have been reported to be more cytolytic in vitro than mononuclear phagocytes obtained from progressing sarcomas (Russell and McIntosh, 1977).

The present investigation was designed to elucidate the effects on in vitro tumor growth of murine

macrophages isolated from a chemically-induced strongly immunogenic, non-metastasizing sarcoma and from a weakly immunogenic, metastasizing line of the same tumor.

MATERIAL AND METHODS Mice

C57BL/6 mice, 8 to 10 weeks old, were obtained from Charles River Breeding Laboratories, Calco, Italy.

Tumors The benzo [alpyrene-induced FS6 sarcoma was

received on three occasions (October 1976, December 1977 and April 1978) from Dr. R. Evans and Dr. S. Eccles, Chester Beatty Research Institute, Sutton, Surrey, England. The tumor does not metastasize and mice immunized by surgical excision of the neoplasm reject up to 4 ~ 1 0 ~ live tumor cells (Parr et al., 1977; Mantovani et al., 1977b). In February 1977, after six passages in this labora- tory a subline, designated mFS6, was isolated from the primary tumor of a mouse with a metastatic nodule in the lung and was maintained by SC passage in syngeneic mice. The mFS sarcoma is weakly immunogenic, 60 and 75% of immunized mice rejecting challenge with lo4 and lo3 tumor cells respectively. The mFS6 and FS6 sarcomas have com- mon tumor-associated transplantation antigens, as detected by cross-resistance in in vivo immunization tests. The mFS6 tumor spontaneously metastasizes to the lung in 45% (range 20-80% in 10 experiments) of IM injected mice. The macrophage content of the FS6 and mFS6 tumors was 44% (56-36, range of eight experiments) and 19% (25-1 1) respectively, assessed by the method originally described by Evans (1972). Similar data were obtained when the number of infiltrating host mononuclear phago- cytes was evaluated in terms of Fc receptor-bearing cells (Pross and Kerbel, 1976). The median survival time of mice transplanted IM with lo5 tumor cells was 38 days for FS6 and 30.5 days for the mFS6 tumor.

Macrophages Macrophage monolayers were prepared in the

wells of Costar trays (3524 Costar, Cambridge,

Received : June 19, I978 and in revised form September 18, 1978.

Abbreviations: MSV, Moloney sarcoma virus; BME, basal medium Eagle: [SH]TdR, [SH]thymidine; A:T, attacker to target cell ratio.

742 MANTOVANI

TABLE I

EFFECTS ON I N VITRO TUMOR GROWTH OF MACROPHAGES ISOLATED FROM THE FS6 A N D mFS6 SARCOMAS ~~

% inhibition ( - - ) or stimulation (+) of PHITdR uptake Macrophage4 obtained from

FS6 mFS6 TLX9 SL2 E

Normal peritoneal cavity +15 t 6 +2512* + 2 8 1 2 * + 1 9 t l * t 2 1 tl* FS6 sarcoma -48 1 3 * -58+1* -45*0.5* -51 t2* -75+3* mFS6 sarcoma -1763 I * $38 t l * +52&3* + 3 5 + 1 * +98_t l*

* p<0.05.

Mass.) essentially as described by Evans (1973). Briefly, tumors obtained 2-3 weeks after implan- tation, unless otherwise specified, were minced with scissors and disaggregated by exposure for 30 min to 0.1 % trypsin or 0.3% crude collagenase (Sigma Chemical Co., St. Louis, Mo.) in basal medium Eagle (BME) containing 10 pg/ml DNase. The percentage of adherent, spreading-out cells was calculated in a hemocytometer (Evans, 1972). The cells were centrifuged and resuspended in BME and approximately 5 x 1 0 macrophages in 1 ml were seeded in the wells and incubated for 30-60 min at 37" C. The wells were then washed thoroughly with jets of medium; >95% of the adherent cells were macrophages as assessed by morphology, phagocytosis of latex particles, avid uptake of neutral red, and binding and phagocytosis of antibody-coated sheep erythrocytes.

TABLE 11

EFFECTS ON I N VITRO TUMOR GROWTH OF MACROPHAGES FROM THE FS6 AND mFS6 TUMORS

~~

% growth inhibition (- Macrophages or stimulation (+) ' obtained from

mFS6 E

Normal peritoneal cavity -15 &6 -18i5 FS6 sarcoma -49*4* -58f3* mFS6 sarcoma +3513* + 5 7 1 5 *

Growth inhibition assay The cytotoxicity assay employed in these studies

has been described previously (Mantovani, 1977 ; Mantovani et al., 1977a, b). Briefly, target cells (5 x104 in 1 ml growth medium) were seeded on macrophage monolayers immediately after the wells were washed and the cultures were incubated for 24 to 48 h at 37" C in air with 5 % C02. Growth of tumor cells was checked daily under an inverted microscope. When the FS6, mFS6 and E adherent target cells were used, the medium was discarded after incubation and the samples were incubated for a further 4-6 h with 1 ml fresh growth medium containing 0.5 pCi [SH]thymidine, [SH]TdR, specific

TABLE I11

EFFECT ON I N VITRO TUMOR GROWTH OF TUMOR- ASSOCIATED MACROPHAGES ISOLATED AT DIFFERENT

TIMFS AFTER IMPLANTATION OF THE FS6 AND mFS6 SARCOMAS

% inhibition (-) or stimulation (+ )

cell DNA synthesis

Day after tumnr transplant of mFS6 tumor Tumor

FS6 15 -52*2* 21 -59*2* 30 -45*4*

mFS6 10 17

' Growth inhibition or stimulation assessed microscopically using mFS6 and E target cells.

* p<O.OS.

* o.rO.05.

24 30

+95&2* +85il* +7914* i- 83 + 5*

Target cells Tumor cells used as targets were: FS6 and mFS6

sarcomas; SL2 and TLX9 lymphomas of DBA/2 and C57BL/6 origin respectively; and the human E cell line, originally derived from an ovarian carci- noma, obtained through the courtesy of Dr. L. Morasca at this Institute. Tumor cells were main- tained in medium RPMI 1640 (Eurobio, Paris, France) supplemented with 10% fetal bovine serum (Gibco-Biocult, Glasgow, Scotland), and 50 ,ug/ml gentamycin (growth medium). TLX9 and SL2 cells grew in suspension whereas FS6 and E cells adhered to plastic and were collected by exposure for 5 min to 0.1 % trypsin and 0.02 % EDTA.

activity 1.9 Ci/mmole, Schwarz Mann, Orangeburg, N.Y. When lymphoma cells growing in suspension were used as targets, the cells were resuspended, transferred to plastic tubes and washed prior to pulsing, as previously described (Mantovani, 1977 ; Mantovani et al., 1977a, b) . Percentage inhibition or stimulation of [3H]TdR uptake was calculated as (1 -A/B) x 100, where A is the isotope uptake in the presence of tumor-associated macrophages and B is the isotope uptake by tumor cells alone. Isotope uptake by macrophages alone was negligible (<500cpm) and was not taken into account in the calculations. In a few experiments, growth

CYTOTOXICITY OF TUMOR MACROPHAGES 743

‘01 Control

0 FS6 macrophages .I

/

0 P’

i 8 72 2‘ hours

FIGURE 1 - Effect of FS6 macrophages on the in vitro growth of mFS6 target cells.

inhibition was evaluated by microscopically counting the number of tumor cells with the aid of an eyegrid after staining with Giemsa. The same formula shown above for [SH]TdR experiments was used to measure growth inhibition, A and B in this case indicating the number of tumor cells in test and control cultures respectively. Under these conditions, normal unstimulated peritoneal C57BL/6 macrophages either do not consistently affect growth and DNA synthesis of tumor cells in vitro or else occasionally slightly enhance [SH]TdR uptake (Mantovani, 1977), Results are presented as mean l f s ~ of three replicates per experimental group. Statistical significance was assessed by Student’s t-test.

RESULTS

Table I shows experiments illustrating the effect on in vitro tumor growth of macrophages from mFS6 and FS6 sarcomas. Peritoneal macrophages isolated from normal C57BL/6 mice either failed to affect or, as in the experiments shown in Table I, slightly enhanced tumor proliferative capacity. Macrophages tested immediately on isolation from the FS6 sarcoma non-specifically inhibited [SH]TdR uptake in a variety of target cells. Similar inhibition of tumor-cell proliferative capacity was observed in a series of experiments in which cytostasis was evaluated in terms of tumor-cell counts (Table 11). In contrast, macrophages from the mFS6 tumor signifi- cantly enhanced tumor-cell proliferative capacity, the effect being totally non-specific (Tables I and 11).

The cytotoxic and stimulatory activities of FS6 and mFS6 macrophages, respectively, were consistently observed with macrophages collected at different times after tumor implantation (Table 111) or when target cells were exposed to macrophages for different times in vitro (Fig. 1). However, the time- course of the growth-enhancing effect of mFS6

macrophages gave a variable pattern of stimulation. In some experiments (3/6) the longer the incubation time the better the stimulatory effect, as illustrated in Figure 2a, whereas in others (Fig. 2b) enhance- ment of tumor-proliferative capacity was best observed after 24 h and tended to decline there- after. The mechanismrs) responsible for the incon- sistency of the time-course of the enhancing effect remains to be elucidated.

The effect on tumor growth of macrophages obtained from the FS6 and mFS6 sarcomas was investigated using serial two-fold dilutions of effector cells. As shown in Figure 3, FS6 macro- phages only showed growth-inhibitory capacity when attacker: target cell (A:T) ratios of 1O:l or 5 : I were employed, lower ratios resulting in no effect (2:l) or stimulation (1 : I ) of [3H]TdR uptake. Optimal stimulation of tumor cell proliferative capacity was detected with 10 mFS6 macrophages per target cell, lower A:T cell ratios causing less enhancement. Similar data were obtained in exper-

26 LB 72 96 0’ h o u r s

Control

0 mFS6 macrophages

1 I

2 4 hours 48 72

FIGURE 2 - Effect of mFS6 macrophages on the in vitro growth of mFS6 target cells.

* F56 macrophages

o mFS6macrophages

MANTOVAN1

10:1 5:l 2 ‘ 1 1:) A T ratio

FIGURE 3 - Effects of tumor-associated macrophages on the in vitro growth of mFS6 target cells tested at different attacker: target cell ratios.

iments in which the A:T ratio was decreased using serial two-fold dilutions of E target cells.

It has been previously described that, while macrophages isolated from certain rat tumors main- tain their cytotoxic potential when cultured, tumor- associated macrophages from other rat or mouse neoplasms rapidly lose their cytotoxicity when maintained in vitro (Evans, 1976). It was therefore of interest to investigate the effect of culture on the growth-inhibitory capacity of FS6 and the stimulatory effect of mFS6 macrophages. As shown in Figure 4, FS6 tumor-associated macrophages rapidly lost their cytotoxic potential when cultured and, indeed, after 24 h significantly enhanced tumor- proliferative capacity. Macrophages from the mFS6 sarcoma maintained their growth-siimulatory capacity when maintained in vitro over a period of 72 h.

DISCUSSION

The results presented here show that macrophages isolated from the immunogenic, non-metastasizing FS6 sarcoma were non-specifically cytotoxic in vitro on tumor cells whereas macrophages from the weakly immunogenic, metastasizing mFS6 line derived from the same tumor significantly enhanced tumor-proliferative capacity.

It has been reported that macrophages infiltrating chemically- or virally-induced rodent tumors can be cytotoxic in vitro on tumor cells, and as shown here with the FS6 sarcoma, in most systems inves- tigated cytotoxicity is non-specific (Evans, 1976; Holden et al., 1976; Russell and Mclntosh, 1977). In the present study, cytotoxic FS6 macrophages rapidly lost their cytotoxic potential after culture and significantly enhanced tumor-proliferative

capacity. A similar loss of cytotoxic activity ‘Bfter culture has been described in various mouse and rat tumors (Evans, 1976; Russell et al., 1977). However, macrophages from certain rat sarcomas maintained their cytotoxic potential for 48 h in culture (Evans, 1976), although the reason for this different behavior is not clear.

In the present study, macrophages tested immedi- ately after isolation from the metastasizing mFS6 sarcoma significantly enhanced tumor-proliferative capacity in vitro non-specifically. Similarly FS6 macrophages stimulated [SH]TdR uptake by target cells after in vitro culture for 24 or 48 h. Normal peritoheal macrophages have been shown to enhance growth of lymphoid tumors which by themselves grew poorly in tissue culture (Nathan and Terry, 1975; Namba and Hanaoka, 1972; Hewlett et al., 1977). Olivotto and Bomford (1974) reported that, after culture, peritoneal mouse macrophages en- hanced DNA synthesis of tumor target cells and Krahenbuhl et al. (1976) showed that, after sub- cutaneous treatment with Corynebacterium parvum or injection of killed Toxoplasma gondii, peritoneal macrophages significantly enhanced [SH]TdR up- take by the L 929 line when compared to untreated effector cells. Proteose peptone-activated macro- phages reportedly enhanced tumor cell DNA syn- thesis at low effector to target cell ratios (Keller, 1973). Evans, using macrophages from chemically- induced sarcomas, showed that after culture they could promote growth of tumor cells, an effect mediated by soluble factors and best observed with target cells in suboptimal culture conditions (Evans, 1976). Recently we observed stimulation of in vitro tumor cell proliferation with macrophage prep-

1 F S 6 macrophages 0 rnFS6 macrophages

/ /

7 2 2 4 hours 0

FIGURE 4 - Effect of culture on the cytotoxic activity of tumor-associated macrophages on mFS6 target cells.

CYTOTOXICITY OF TUMOR MACROPHAGES 745

arations from human ascitic ovarian tumors (Mantovani et af., submitted for publication). Thus, there are scattered reports of stimulation of in vitro tumor growth by macrophages and we demonstrate a similar effect using macrophages tested immedi- ately after isolation from a spontaneously meta- stasizing murine sarcoma. The in vivo significance of the tumor-growth-promoting effect of tumor- associated ’bacrophages and its possible relevance in the context of an immunostimulation theory of tumor development (Prehn, 1977) remain to be elucidated. It is of interest in this connection that, in the poorly immunogenic metastasizing Lewis lung carcinoma model, the anti-macrophage agents silica (Sadler et al., 1977) and carrageenan (Mantovani and Polentarutti, unpublished data) inhibit primary tumor growth in vivo while stimu- lating metastasis.

In the present investigation, a correlation was observed between tumor immunogenicity and meta- stasizing capacity, on the one hand, and cytotoxicity of tumor-associated macrophages, on the other. Similarly, in the mouse MSV system, Russell and co-workers reported that macrophages obtained from regressing sarcomas were more cytotoxic

in vitro than mononuclear phagocytes isolated from progressing tumors (Russell and McIntosh, 1977). On the other hand, Evans tested the in vitro growth-inhibitory capacity of macrophages infil- trating a series of rat chemically-induced sarcomas after culturing effector cells for 24 to 48 h, and found no correlation between in vitro cytotoxicity and in vivo behavior of the tumors examined (Evans, 1976). The different animal species and the culturing of macrophages prior to testing might partially account for the apparent discrepancy between data obtained from mouse and rat tumors.

The in vivo significance of the in vitro effects of tumor-associated macrophages remains a matter of speculation. The correlation reported here between in vitrokffects of macrophages and in vivo metastasizing capacity appears in keeping with the hypothesis tlwt mononuclear phagocytes may play a role in the regulation of tumor growth and metastasis.

ACKNOWLEDGEMENT

This work was supported by Grant No. ROI- CA-12764 from the National Cancer Institute, Bethesda, Md., USA.

EFFETS SUR LA CROISSANCE TUMORALE I N V I T R O DES MACROPHAGES MURINS ISOLES A PARTlR DE LIGNEES DE SARCOME DIFFERENTES AUX POINTS DE VUE IMMUNOGl?NICITE

ET CAPACITE METASTATISANTE Des macrophages ont et6 isolts B partir d’un sarcome FS6 - tumeur fortement immunogenique et ne produisant pas

de metastases - transplant6 chez des souris C57BL/6 syngeniques, et d’une lign6e (mFS6) de la m&me tumeur, mais faible- ment immunogenique et qui se metastatise spontan6ment. Les macrophages provenant du sarcome FS6 inhibaient non sp6- cifiquement la synthkse du DNA dans les cellules tumorales ainsi que leur croissance in vitro. En culture, ils perdaient rapidement leur activite cytotoxique et devenaient capables de stimuler non specifiquement la croissance tumorale in vitro. Les macrophages de la lign6e mFS6 augmentaient non spkifiquement I’activitk proliferative des cellules cibles tumorales; cette capacite persistait en culture.

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