PART 11. IMMUNE RESPONSES TO TUMORS AND THEIR ANTIGENS: CELLULAR AND HUMORAL MECHANISMS

THE SIGNIFICANCE OF MACROPHAGES IN HUMAN AND EXPERIMENTAL TUMORS *

Peter Alexander, Suzanne A. Eccles, and C. L. L. Gauci

Chester Beatty Research Institute Belmont, Sutton, Surrey, England

Introduction

Macrophages are involved at several stages in the genesis of an immune response to tumors. They process antigens, they are required in the transforma- tion by tumor antigens of memory lymphocytes into cytotoxic lymphocytes, and they are also important effector cells that can be both specifically cytotoxic for cells bearing a particular tumor antigen as well as generally tumoricidal.’ The in vivo relevance of macrophage cytotoxicity in the tumor-host relationship remains to be established.

A tumor is a complex structure consisting of malignant cells supported by a stroma and a vascular system, both of which are made up of normal cells. In addition, there are also present normal inflammatory cells such as lympho- cytes, polymorphonuclear leucocytes and macrophages. It is a serious error to equate “cells in a tumor” with “tumor cells’’; the latter can be in the minority. In this paper we deal with the macrophage content of tumors and its possible relation to the biological properties of the tumor. It must be remembered that macrophages are only one of several nonmalignant cell types that constitute an integral part of a tumor.

Measurement of the Macrophage Content of Tumors

The presence of macrophages, both at the periphery and within neoplasms, is well recognized; and in the case of melanomas, macrophages that have in- gested pigment may be mistaken under light microscopy for tumor cells. Evans demonstrated that some experimental rodent sarcomas contain large numbers of macrophages, which are not readily recognized in histological sections by light microscopy. He further showed that the macrophages in these transplanted tumors were derived from the host and not from the tumor implant. Cells in the tumor were shown to be macrophages by dispersing the sarcomas with hydrolytic enzyme and then studying the cell suspension obtained. Macrophages unlike sarcoma cells spread and adhere to glass or plastic in the presence of trypsin. That cells showing this behavior were largely macrophages and that the cells remaining in suspension contain almost no macrophages was shown by the capacity of the adherent cells to phagocytose several opsinized sheep red cells and to bind to their surface a highly specific antimacrophage serum.

The procedure used for estimating the number of macrophages in human

+ This work has been supported by Grants from the Medical Research Council and the Leukaernia Research Fund. C.L.L. Gauci is in receipt of a Cancer Research Campaign Fellowship.

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Alexander et al. : Significance of Macrophages 125

tumors was similar to that employed for rodent tumors and relied on obtaining a representative cell suspension. A key reagent in these investigations is a rabbit anti-human macrophage serum that binds only to the cell membrane of cells belonging to the mononuclear phagocyte series (including promonocytes) .

Factors That Determine the Macrophage Content of Rat Sarcomata

Evans found that the macrophage content of a series of different, chemi- cally-induced rat sarcomas either autochthonous or syngeneic transplants, ranged from 3 to 58% of the total cell population of the tumor. The variations in the macrophage content of tumors may be due: ( 1 ) to differences in the struc- ture of the blood vessels within these tumors (in that extravasation through the blood vessels into the extravascular spaces of the tumors may be different); or (2) the macrophages (or monocytes) may enter the tumor as part of an immune reaction of the host against the tumor by mechanisms similar to the

TABLE 1

TRANSPLANTED RAT SARCOMA EFFECT OF DEPLETION OF T LYMPHOCYTES ON THE MACROPHAGE CONTENT OF A

Macrophage Content

of Tumor* (% ) Treatment Prior to Implantation of Tumor None 54 ( 2 4 ) Adult thymectomy: 3 x 300 R x rays; plus 4 weeks to

allow marrow recovery 21 ( + 3 ) Sham thymectomy: 3 x 300 R x rays; plus 4 weeks recovery 51 ( + 5 ) 8 days draining of thoracic duct lymph 31 (+9) Sham cannulation 52 (+4)

*Intramuscular growth for 14 days when tumors were approximately 2 cm in diameter in all groups.

entry of monocytes into sites involved in a delayed hypersensitivity response. In the experimental tumors studied by us,' the second mechanism appears to be the most important because, as is shown in TABLE 1, the macrophage con- tents of tumors is reduced if these are transplanted into hosts that are deficient in T cells. In these experiments, two different procedures were used to render animals deficient in T cells so that they can accept allografts and yet have a normally functioning bone marrow. That the bone marrow of the T-cell- deprived animals was normal, at least as far as the reduction of monocytes is concerned, was found from comparison of the number of monocytes that appear in inflammatory exudates-this was the same in the T-cell-deprived animals as in normal animals5 Consequently, the decrease in the number of macrophages in tumors grown in T-cell-deprived animals cannot be due to nonavailability of circulating monocytes and must be attributed to the defect in specific cell- mediated immunity that is evidenced by the fact that these rats accept allografts.

The hypothesis that macrophages enter the tumor as part of a specific reac- tion of the host directed against tumor-specific antigens is supported by the

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observation that the macrophage content of tumors is directly related to the “immunogenicity” of the sarcomas used. The measure for “immunogenicity” is the number of tumor cells a rat is capable of rejecting following immunization (e.g. by surgical removal of a prior transplant of the same tumor). Those sarcomas that produced high specific resistance by this test contained the highest number of macrophages, whereas sarcomas that were essentially “nonimmuno- genic” (i.e., their prior immunization did not lead to increased resistance to a subsequent challenge) contained almost no macrophages. It must be stressed that “immunogenicity” as measured by graft rejection is not synonymous with antigenicity. Immunogenicity is a complex parameter that involves both the magnitude of the host response evoked by the tumor-specific antigens, and the capacity of the injected cells to evade destruction by the effector mechanisms, humoral and cell-mediated, produced within the immunized (or tumor-bearing) host. We have found that some rat sarcomas that are “nonimmunogenic” can yet be antigenic when this is assayed by determining the specific cytotoxicity in vitro of the lymphocytes in the nodes draining the tumor. In this situation, we interpret lack of “immunogenicity” as being due to a very efficient escape mechanism by the sarcoma ce11s.O. 7

Annals New York Academy of Sciences

Metastatic Behavior and the Macrophage Content of Rat Tumors

The capacity of different experimentally induced rat sarcomas, when trans- planted into syngeneic hosts, to give rise to distant metatases was determined by implanting the tumor intramuscularly and then removing it and the draining node 14 days later. The animals were then kept for several months and the incidence of distant metastases scored.4 TABLE 2 shows that there is an impres- sive inverse correlation between the capacity to metastasize and the macrophage content of the different tumors studied. We also observed that tumors which have a high macrophage content and which when transplanted into normal animals do not metastasize significantly, give rise to a high incidence of metas- tasis when transplanted into T-cell-deprived rats, when the macrophage content as shown in TABLE 1 is much reduced.

Monocytosis in an Acute Myeloid Leukemia of Rats

A transplantable leukemia that arose spontaneously was found to have many of the characteristics of acute myeloblastic leukemia. In syngeneic recipients, as few as ten cells give rise to leukemia, which rapidly disseminates to the bone marrow and blood, and at the time of death there are more than lo5 leukemic blast cells/mm3 of blood. As the number of leukemic blast cells rises in the blood following transplantation of this tumor, so does the number of monocytes, and one day before the death of the animal the number of monocytes in the blood is increased more than 20 times over that in normal rats.* By trans- planting the leukemia into F, recipients (August x Marshall rats-the leukemia is of August phenotype) it could be shown conclusively that the monocytes were of host origin and not derived from the leukemia cells. The blood leucocytes were cultured when the monocytes adhered to glass and turned into macro- phages. These were then exposed to an alloantiserum directed against trans- plantation antigens of the Marshall phenotype. The macrophages derived from

Alexander et nl. : Significance of Macrophages 127

the blood monocytes were all of the F, (August X Marshall) phenotype, whereas the nonadherent leukemic blast cells were invariably of the August phenotype. Also, when the leukemia was grown in rats in which the bone marrow had been compromised, the number of monocytes was greatly decreased.

The monocytosis induced by the leukemia is, however, a different plie- nomena from the entry of macrophages into immunogenic sarcomata because this leukemia is only very weakly immunogenic. Also, this monocytosis is equally great when the leukemia is transplanted into T-cell-deprived rats and, as will be shown in the next section, the monocytes in the blood of the leukemic rat have normal functions, whereas the monocytes in the blood of rats bearing highly immunogenic tumors are abnormal in that they do not enter sites of inflammation. The investigations with the leukemia emphasize that the stimula- tion of the bone marrow that accompanies the growth of some tumors such as this leukemia, is a different phenomenon from the entry of large numbers of

TABLE 2 RELATIONSHIP BETWEEN THE MACROPHAGE CONTENT AND CAPACITY TO CAUSE DISTANT METASTASIS WITHIN ONE YEAR OF REMOVAL OF PRIMARY TRANSPLANT FOR A SERIES OF CHEMICALLY-INDUCED RAT SARCOMAS TRANSPLANTED INTO

SYNCENEIC RECIPIENTS.

Macrophage Content % Incidence of Tumor Mean Range Metastasis * Immunogenicity t

MC-3 8 2-12 . 100 < 1o-q HSH 12 10-15 100 10' ASBP-1 22 18-26 52 l@ MC-l(M) 38 26-42 25 1V HSN 40 34-44 32 5 X l V HSBPA 54 42-63 1 1 5x10'

;: Following amputation of tumor-bearing limb (i.e. removal of local tumor plus

t Number of cells needed to induce an i.m. tumor in rats immunized by excision of its draining nodes) 14 days after inoculation of tumor cells.

im. tumor.

monocytes into a solid sarcoma. The latter is determined by the immune reac- tion of the host against the tumor, whereas the former appears not to be related to an immune response.

Sarcomas Interfere with the Entry of Monocytes into Sites of Inflammation

Rats bearing transplanted syngeneic sarcomata have a reduced capacity to mount an inflammatory reaction and a delayed hypersensitivity response to unrelated antigems FIGURE 1 shows that the entry of monocytes into the peritoneal cavity 72 hours after the introduction of the irritant oyster glycogen is reduced in rats bearing sarcomas. The impairment increases progressively as the tumor grows, and its magnitude is related to the macrophage content of the tumor. It is greatest for tumors that have the highest macrophage content.

128 Annals New York Academy of Sciences

Similarly, the capacity of rats that have been immunized with BCG to mount a delayed hypersensitivity reaction (as measured by footpad swelling) to chal- lenge with P.P.D. is greatly reduced in rats bearing tumors, (see TABLE 3 and FIGURE 2). The reduction in the ability to mount a delayed inflammatory response, both when this is induced by an antigen and by an irritant, is directly related to the size and macrophage content of the sarcoma (see FIGURE 2).

The decrease in the number of monocytes that enter the sites of inflamma- tion (i.e., the peritoneal cavity in response to oyster glycogen) is not due to a reduction in the number of circulating monocytes and, indeed, rats bearing

I I I I

10 20 30 Tumaur weight. (grams)

FIGURE 1. Effect of growing tumor on the number of macrophages that are in- duced into the peritoneal cavity 72 hours after stimulation with oyster glycogen. In control rats the number of peritoneal macrophages is increased from 0.2 x lo' to 1 . 4 ~ 10' as a result of injecting oyster glycogen. In tumor-bearing rats the resident population of macrophages is within the normal range but the number that can be induced by oyster glycogen is reduced. The extent of the reduction increases with tumor weight and is greatest for tumors containing most macrophages. - Num- ber of macrophages following oyster glycogen; - - - - - - number of macrophages resi- dent in the unstimulated peritoneal cavity.

sarcomas have a monocytosis and not a monocytopenia.D As a result of the presence of a tumor, the properties of the circulating monocytes are modified in such a way that they do not enter into inflammatory sites. This can come about because in the tumor-bearing host the monocytes lose the capacity to extravasate and/or respond to chemotactic stimuli. It is possible that this im- pairment is caused by circulating antigen-antibody complexes that have been shown to be present in rats bearing tumors having a high macrophage content.1° Antigen-antibody complexes bind to Fc receptors, and this interaction may modify the behavior of the monocytes.

The very pronounced monocytosis seen in rats with acute leukemia is un-

Alexander et al. : Significance of Macrophages 129

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Different primary and transplanted sarcomas: all approximately 2cm in diameter.

0 A o 0

dd 10 20 30 LO 50 60 70 ae

K Macrophages in turnour

FIGURE 2. Suppression of delayed hypersensitivity reaction in rats immune to BCG and challenged in the footpad with P.P.D. is directly related to macrophage content of tumor. 0 different autochthonous benzpyrene-induced tumors; A different trans- planted benzpyrene-induced tumors; 0 different transplanted methylcholanthrene in- duced tumors.

TABLE 3 REDUCED CAPACITY OF TUMOR-BEARING RATS TO MOUNT A DELAYED CUTANEOUS

HYPERSENSITIVITY REACTION TO B.C.G.

% Increase in Foot Thickness 24 hours after P.P.D.

Tumor Days after Days after Inoculated Tumor Inoculation Tumor Excision

7 14 21 1 3 7

None 33.025.9 28.8k4.2 30.725.0 25.9k6.2 32.523.1 31.824.8 (controls) MC3 (low 30.05k3.1 19.0k4.1 12.4021.4 N.T. N.T. 29.8k4.8 macrophage content HSBPA (high 23 .2k 1.8 10.122.2 1.520.8 16.5k8.4 17.9k5.3 28.623.2 macrophage content)

* The rats were immunized with BCG and challenged with PPD 13 days later. The impairment increases progressively as the tumor grows but is reversed rapidly following excision of the tumor. The magnitude of the effect is greatest with sarcomas with a high macrophage content.

130 Annals New York Academy of Sciences

related to an immune response, and it is of interest that rats with the acute leukemia mount normal inflammatory responses both in terms of the delayed hypersensitivity reaction as measured by footpad swelling and the response to the nonspecific stimulus, i.e., the injection of oyster glycogen into the peritoneal cavity.8 We conclude that the blood monocytes in rats with the SAL leukemia function normally when confronted with an inflammatory situation. This obser- vation is consistent with the hypothesis that the defect in the blood monocytes in rats bearing tumors with a high macrophage content is caused by circulating antigen-antibody complexes. In rats with this leukemia, which is only very weakly immunogenic, the concentration of antigen-antibody complexes is likely to be low.

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Macrophage Content of Some Human Tumors

In a preliminary communication,3 the macrophage content-assayed as described above-of surgical specimens of human tumors was found to vary widely. FIGURES 3 and 4 summarize our total experience to date on the macro- phage content of human tumors; 54 malignant and 13 benign lesions have been investigated.

FIGURE 3 shows the findings for breast tumors and melanomas. It is note- worthy that all of the benign breast lesions had a low content of macrophages,

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FIGURE 3. Macrophage content in surgical specimens of breast tumors and mela- nomas.

Alexander et al. : Significance of Macrophages 131

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FIGURE 4. Macrophage content in surgical specimens of 54 malignant tumors in: (a) primaries with no clinical evidence of dissemination; (b) local recurrences with no clinical evidence of dissemination; (c) distant metastatic lesions removed some time after surgical removal of the primary. (Breast carcinoma 17; melanomas 25; squamous cell carcinomas 6; sarcomas 2; g.i. tract adenocarcinomas 3; thyroid car- cinoma 1).

and in this group the four tumors with the greatest numbers of macrophages showed inflammatory changes on histological examination. A possible interpre- tation is that the cells giving rise to the benign lesions are not antigenic. The malignant breast lesions fell into two clinical classes: those that at the time of surgery showed no evidence of local or distant dissemination, and those that at the time of surgery had invaded local nodes or skin. The primary lesions that had given rise to skin or node involvement all contained few macrophages, whereas the primary lesions of those tumors that were clinically localized at the time of surgery had a macrophage content which ranged from 0 to 30%. The follow-up has not been sufficiently long to relate prognosis with the macrophage content of the malignant lesions from Stage 1 breast cancer patients with prognosis.

In the case of the melanomas, all of the metastatic tumors had a low macro- phage content, whereas tumors removed as primaries or as local recurrences (all of the local recurrences were cases for which there was at surgery no clinical indication of distant dissemination) contained between 9 and 30% macro- phages. As in the case of the patients with breast cancer, we shall have to wait several years to know whether the macrophage content of primary or locally recurrent malignant melanomas is of prognostic significance. One reason for believing that this may be the case is the finding that metastatic lesions invariably

132 Annals New York Academy of Sciences

have less numbers of macrophages. This is brought out in FIGURE 4, in which the macrophage content of 54 malignant lesions of a wide variety of histological types is subdivided into three groups: One, primary tumors, for which at the time of surgery there was no indication of dissemination; two, locally recurrent tumors with no evidence of distant spread at surgery; and three, metastases, which were distant from a primary tumor that had been removed earlier. It is evident that all of the metastatic lesions contained low numbers of macrophages, whereas the macrophage content of primary lesions varied from 0 to 35%.

In rats with transplantable sarcomas the macrophage content of the “pri- mary” (i.e. the tumor produced at the site of the inoculation of the tumor cells) and that of the distant metastases that developed after surgical removal of the tumor (Eccles, unpublished) were very similar. While “primaries” with high macrophage content gave rise to few metastases, those that occurred had a high macrophage content. On the other hand, the frequently occurring metastases in rats that had “primaries” with low macrophage content contained few macro- phages. We shall have to wait several years to know if the pattern in man is similar. As yet, we have had no opportunity to examine both a primary and a distant metastasis from the same patient.

CONCLUSIONS

The macrophage content of human and experimental tumors varies widely. In rats, it is clear that the presence of large numbers of macrophages within a solid sarcoma is a favorable prognostic indicator (once the tumor has been excised), as such tumors metastasize less readily than those with a lower macro- phage content. The entry of macrophages into the rat sarcomas is associated with an immune reaction of the host to the tumor and it is probably this immune reaction which is responsible for restraining metastatic ~ p r e a d . ~ Whether the macrophages within the tumor contribute to the immune control of the tumor or whether they are irrelevant bystanders attracted into the tumor by lympho- kines or chemotactic factors released when immune lymphocytes or antibodies interact with the tumor is not known, because studies on the cytotoxicity of tumor macrophages have so far been inconclusive.’ A definitive answer could come from experiments with animals that are depleted of monocytes and macro- phages but have normal lymphocytes. As yet, it has not been possible to produce such animals. Administration of substances that selectively kill macrophages, such as silica or antimacrophage serum, are relatively ineffective in reducing the macrophage content of tumors (Eccles, unpublished). This may be because extensive depletion of macrophages cannot be achieved without killing the animals.

In man, the prognostic significance of the macrophage contents of tumors remains to be established. All we know is that different tumors of the same histological type vary widely in the number of macrophages they contain.

The sequestration of large numbers of macrophages within tumors appears to be a different phenomenon from the monocytosis induced by tumors.9 The latter is probably the result of direct stimulation of the bone marrow by tumor growth and need not relate to the immune response of the host to the tumor. In rats bearing highly immunogenic sarcomas, the number of monocytes that enter inflammatory lesions is greatly reduced in spite of the fact that there is a monocytosis. On the other hand, a transplantable leukemia induced a mono-

Alexander et al. : Significance of Macrophages 133

cytosis in which the monocytes have the normal capacity to enter inflammatory lesions.

The following three phenomena must be distinguished: One, the entry of monocytes into tumors to give rise to tumors with a high macrophage content; two, the monocytosis induced by tumor growth; and three, the defect of circu- lating monocytes in rats with immunogenic sarcomas which stops their entry into sites of inflammation.

REFERENCES

1. LEVY, M. H. & E. F. WHEELOCK. 1974. Adv. Cancer Res. 18: 131-163. 2. EVANS, R. 1972. Transplantation 14: 468-473. 3 . GAUCI, C. L. & P. ALEXANDER. 1975. Cancer Letters 1: 29-32. 4. ECCLES, S. A. & P. ALEXANDER. 1974. Nature 250: 667-669. 5. ECCLES, S. A. & P. ALEXANDER. 1974. Br. J . Cancer 30 42-49. 6. CURRIE, G. A. & P. ALEXANDER. 1974. Br. J . Cancer, 29: 72-75. 7. ALEXANDER, P. 1974. Cancer Res. 34: 2077-2082. 8. GAUCI, C. L., A. WRATHMELL & P. ALEXANDER. 1975. Cancer Letters 1: 33-37. 9. ECCLES, S. A., G. BANDLOW & P. ALEXANDER. Br. J . Cancer. In press.

10. THOMSON, D. M. P., S. A. ECCLES & P. ALEXANDER. 1973. Br. J. Cancer 28: 6-15.