Macrophage-mediated cytostasis, unlike cytotoxicity, is ex- hibited against both neoplastic and normal target cells (2,7) as well as proliferating normal lymphocytes (7,8,9)

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CYTOSTASIS OF TUMOR AND NONTUMOR CELLS

Alan M. Kaplan

INTRODUCTION

In vitro inhibition of tumor growth (cytostasis) and tumor cytolysis (cytotoxicity) have been shown to be mediated by T cells, K cells, natural killer cells, and macrophages (M0)

[reviewed in (1)]. Cytostasis mediated by activated macrophages (ΑΜ0) has been demonstrated by reduced cell counts of target cells, reduced target cell clonability, inhibition of mitosis, and by the reduced ability of target cells to incor- porate [*%]- or t¹^]-labeled nucleosides into DNA (2-6).

Macrophage-mediated cytostasis, unlike cytotoxicity, is ex- hibited against both neoplastic and normal target cells (2,7) as well as proliferating normal lymphocytes (7,8,9). While soluble factors have been implicated in M0-mediated cytostasis, most reports have suggested that cell-to-cell contact is a prerequisite for cytostasis to occur (10).

The implication that interaction of ΑΜ0 with target cells results in inhibition of proliferation is of great potential

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consequence, not only in tumor immunology but in cell biology in general. With respect to M0, recent data suggest that growth regulatory properties extend to immunologically unre- lated systems such as wound healing (11). Unfortunately, tumor cell growth as measured either by actual cell counts or isotope incorporation is subject to a series of culture con- ditions including vessel shape, specific activity of the radio- labeled nucleoside, degree of activation of the effector cells, release of a series of effector cell products including arginase (12,13) and thymidine (14-17), and cytolytic effects generated during the culture (1). These problems which, for the most part, are ignored have prompted Gyongyossay et al. (1), in a recent article discussing the methodological problems in- herent in cell-mediated cytostasis, to raise the question as to whether or not cell-mediated cytostasis as a distinct in vitro phenomenon exists.

II. REAGENTS

Target cells (Lewis lung carcinoma, mouse embryo fibroblasts, methylcholanthrenë fibrosarcomas, B-16 melanoma, etc.)

Trypsin: EDTA (10X) (GIBCO 610-5405, GIBCO, Grand Island, New York)

Eagle' s minimum essential medium (EMEM) with Earle's balanced salt solution, 2X essential amino acids, vitamins, 100 U penicillin/ml, 100 yg streptomycin/ml and 20% heat-inac- tivated fetal bovine serum

Tissue culture flasks Trypan blue

96-Well microtest plates (Linbro Scientific, Inc., Hamden, Connecticut)

Sterile 4 x 4 in. gauze pads

Hanks¹ balanced salt solution (HBSS)

Brewer's thioglycollate (Difco Laboratories, Detroit, Michigan)

Protease peptone (Difco Laboratories, Detroit, Michigan) Pyran copolymer (lot XA124-177, Hercules, Inc., Wilming- ton, Delaware)

Corynebacterium parvum (Burroughs-Wellcome, Research Triangle, North Carolina)

Glycogen (type II from Oyster, Sigma G-8751, Sigma Chemi- cal Co., St. Louis, Missouri)

Mycybacterium bovis, strain BCG (Phipps substrain TMC No. 1029 (Trudeau Inst., Saranac Lake, New York)

Purified protein derivative (PPD, Connaught Laboratories, Toronto, Canada)¹²⁵Iododexoyuridine (IUdR, >2000 Ci/mmol, NEX-072, New England Nuclear, Boston, Massachusetts)

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5-Fluorodeoxyuridine (FUdR, Hoffman-La Roche, Nutley, New Jersey)

Methanol

Clear lacquer varnish

III. PROCEDURES

A. Target Cell Preparation

Target cells (Lewis lung carcinoma, mouse embryo fibroblasts, methylcholanthrene fibrosarcomas or B-16 melanoma) are trypsinized from tissue culture after growth for two to three days. The cells are usually approximately 90% confluent at the time of use. Cells are trypsinized from 75 cm² culture flasks by pouring off the culture supernatant, washing twice with 5 ml of sterile saline and adding 2.5 ml of 0.25% trypsin- EDTA solution. After incubation at 37°C for 5 to 10 min, the flasks are tapped several times on the bench top to dislodge the cells, and 2.5 ml of complete EMEM is added. The cells are removed from the flasks by repeated pipetting to break up clumps, centrifuged at 200 g, resuspended in complete EMEM, and viability determined by trypan blue dye exclusion. Target cells are diluted to 5 x 10⁴ cells/ml in complete EMEM, 0.2 ml

(10^ cells) added to the wells of a 96-well microtest plate, and the plates incubated at 37°C for 24 hr. Prior to adding effector cells, the medium is dumped from the wells and the plate drained on sterile gauze pads. The wells are washed twice with complete EMEM by gently pipetting 0.2 ml medium down the side of each well, and dumping the medium as before.

B. Peritoneal Cell Preparation

Peritoneal exudate cells (PEC) are obtained from normal or treated mice by washing the peritoneal cavity twice with 4 ml of Hanks' balanced salt solution (HBSS) without serum.

The PEC are pooled in 50 ml sterile plastic centrifuge tubes and collected by centrifugation at 200 g, washed twice with HBSS, and resuspended in complete EMEM. (We do not routinely use heparin in the wash fluid but 10 U preservative-free heparin/ml reduces clotting.) To obtain "elicited" or "activated"

macrophages, mice may be inoculated ip with any of a number of sterile inflammatory agents or immunomodulators. Some typical substances, doses and schedules are given below with respect to time of inoculation prior to harvest

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Saline, sterile pyrogen free, 1.0 ml ip, day -2.

Brewer's thioglycollate, 10%, 1.0 ml ip, day -3 to -5.

Protease peptone, 10%, 1.0 ml ip, day -3 to -5.

Glycogen, 2.5%, 1.0 ml ip, day -3 to -5.

Pyran copolymer, 25 mg/kg in 0.2 ml ip, day -7.

Killed C. parvum, 17.5 - 70 mg/kg in 0.2 ml ip, day -7.

Mycobacterium bovis, strain BCG (Phipps substrain, TMC No. 1029, 5 x 10⁶ colony-forming units (CFU), id day -30 to -90, 5 x 10⁶ CFU ip, day -3 to -7.

Mycobacterium bovis, BCG, 5 x 10⁶ CFU id on day -30 to -90, PPD, 50 yg ip, day -1 to -3.

The washed PEC in complete EMEM are added to the microtest wells to provide ratios of 20:1 (10⁶/ml or 2 x 10⁵/0.2 ml/well),

10:1, 5:1, and 1:1. After 2-hr incubation at 37°C, the non- adherent cells can be removed by washing two to three times as described above. In general, we have found that there is little or no difference in the results obtained when total cells or the adherent cell population are used. Alternatively, any of a number of procedures can be utilized to purify M0 prior to their addition to the microtest plates including purification by adherence to collagen-coated plates and removal by collagenase (18), purification by adherence to FCS-treated plates and removal by EDTA (19), and purification by adherence to parafilm and removal at 4°C (20).

C. Incorporation of¹^^bIUdR

At various times after cocultivation of peritoneal macrophages (PM) and target cells (usually 24 and/or 48 hr), the medium is dumped and the plates washed three times as described above. The isotope¹²⁵IUdR is added at 0.5 yCi/ml in 0.2 ml in complete EMEM containing 5 x 10"^ M FUdR and the plates are incubated at 37°C for 2 hr in 5% C02· After incubation, the medium with¹²^IUdR is dumped and the plates are washed three times as described above. The plates are fixed with methanol for 10 min, allowed to air dry, then sprayed with clear lacquer varnish and allowed to air dry. The plates are cut with a band saw to separate the individual wells and the individual wells are placed in carrier tubes and counted in a gamma scintillation spectrometer. The assay is carried out in quadruplicate and controls include target cells alone, effector cells alone, target cells with resident PM, and target cells with the appro- priate diluent for any agent being tested for induction of cytostatic macrophages.

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IV. CALCULATION OF DATA

The data can most simply be expressed as counts per minute of⁵IUdR incorporated at a given effector:target cell ratio.

Alternatively,

cpm target cells + effector cells

% Inhibition = [1 — — — — ] x 100, cpm target cells + control cells

where control cells are obtained from mice treated with the diluent used for the agent tested for its capacity to induce cytostatic macrophages. This formula assumes that incorporation into effector and control macrophages is negligible and must be modified if this is found not to be the case.

V. CRITICAL COMMENTS

The cytostasis assay as outlined is, for the most part, a straightforward, highly reproducible assay within a given ex- periment. [^H]-Thymidine or²⁵IUdR incorporation as a technique for assessing the relative amount of DNA synthesis has obvious advantages over counting cells in a hemocytometer or an automated counter. The technique is extremely rapid com- pared to visual means, and allows large numbers of samples to be processed in an objective manner.

Several alternatives exist for various steps in the cytostasis assay. Peritoneal exudate M0 can be purified by adherence directly onto the microtest plates before adding the target tumor cells. Cells can be harvested with sodium do- decysulfate after incubation with¹²⁵IUdR and washing and ex- tracted with 10% cold trichloroacetic acid (TCA) to precipi- tate acid insoluble DNA-bound^{1 2 5}i (21). While as much as 25%

of the counts of 125j associated with the tumor cells can be non-TCA precipitable, the results of a given assay are quite similar regardless of whether whole cells or TCA precipitable DNA is counted (4). Lastly, [³H]-thymidine can be used in- stead of¹²⁵IUdR to measure DNA synthesis. The use of¹²⁵IUdR is substantially simpler and less expensive in that preparation for liquid scintillation counting is avoided. Similarly, the plate assay that we have described while overestimating thé cpm¹²^IUdR provides a fast and accurate assessment of relative DNA synthesis among different groups.

Target susceptibility to M0-mediated cytostasis is varied and includes cells of varied species, both lymphoid and non- lymphoid tumors and cells grown in both monolayer and suspen- sion including L-929 (4), EMT-6 mouse adenosarcoma (4), tran- sitional cell bladder tumor 4934 (4), Lewis lung carcinoma

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(3,7), mouse embryo fibroblasts (3,7), lymphocytes (7,9), SL2 lymphoma cells (17), methylcholanthrene and dimethylbenzan- threne-induced fibrosarcomas (5), polyoma virus-induced tumors

(5), rat fibroblasts (2), human melanomas, MEL-1 and RPMI 7932 (2), Burkitt's lymphoma, RAJI (2), human osteogenic sarcoma (2) and human WI-38 lung fibroblasts (3,7). The relative cytostatic activity of various macrophage populations (normal, elicited, activated) differ somewhat from publication to publication perhaps due to the varying background flora from mouse colony to mouse colony. In general, lymphocyte blastogenesis is not severely inhibited by normal macrophages, only weakly inhibited at high normal macrophage-to-lymphocyte ratios and enhanced at low normal macrophage-to-lymphocyte ratios (9,22, 23). Activated mouse macrophages (pyran and C. parvum) inhibit PHA, con A, and LPS-induced lymphocyte blastogenesis (9,23), while elicited (protease peptone) but not normal rat macrophages also inhibit lymphocyte blastogenesis (22). The cytostatic effect of elicited rat peritoneal macrophages for both lymphocytes and tumor cells and the cytocidal activity of elicited rat peritoneal macrophages for tumor cells is in contrast to the mouse system where elicited macrophages are not cytocidal. In the mouse model normal peritoneal macrophages are generally not cytostatic or only weakly cytostatic to tumor and normal target cells while activated macrophages tend to be strongly cytostatic (7,24,25).

A major problem in the cytostasis assay is in intrepreting the data and avoiding the numerous artefacts that either directly or indirectly alter cell growth and, while causing the appearance of cytostasis in the in vitro environment, are un- likely to be relevant in vivo. This is particularly problem- atic when a single technique is used to assess cell proliferation since several laboratories have demonstrated that cell- free supernatants of macrophages inhibit thymidine or¹²⁵IUdR uptake by cells without blocking cell proliferation (14-17).

The factor that is inhibiting thymidine or⁵IUdR incorporation is synthesized by macrophages, has been identified as thymidine, and acts as a cold inhibitor of labeled nucleoside incorporation (15-17). Under normal experimental condi- tions washing the cultures before pulsing with thymidine or IUdR removes the competitive inhibitor and prevents the appar- ent cytostatic effect (17) except in the EL4 system that is uniquely sensitive to thymidine blockade resulting in inhibition of DNA synthesis and cell growth (16).

Another problem frequently overlooked in evaluating cytostasis experiments is the potential cytotoxicity of the effector cells. Activated macrophages that are cytotoxic for tumor cells also appear to be extremely cytostatic, however, if the target cells are dying in culture, the resulting decrease in cell number and/or isotope incorporation would be wrongfully

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interpreted as cytostasis. Cytostasis can be detected earlier after the initiation of cultures (approximately 2 to 6 hr) than cytotoxicity, but it is not known whether or not cytostasis is a prerequisite for cytotoxicity, shares a common mechanism with cytotoxicity, or is irrelevant to cytotoxicity.

Perhaps the ideal target cell for cytostasis assays would be a tumor cell that is resistant to the cytotoxic effects of macrophages or any other effector cell in question; however, it is not clear that such a tumor target cell exists. Inter- estingly, normal cells in culture and lymphocytes are generally susceptible to activated macrophage-mediated cytostasis while being resistant to the cytotoxic effects of macrophages and, therefore, may make good targets for a cytostasis assay.

However, the relevance of the cytostatic activity of activated macrophages or any other effector cell for normal targets is of questionable in vivo significance.

Acknowledgment

Supported in part by American Cancer Society Grant

#IM183 and NIH Grant #CA28308.

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