[CANCER RESEARCH 42. 3851-3857, September 1982]0008-5472/82/0042-0000$02.00

Tumoricidal Effect of Macrophages Exposed to Adriamycin in Vivo or in V/tro1

FrançoisMartin, Anne Caignard, Olivier Olsson, Jean FrançoisJeannin, and Annick Leclerc

Research Group on Digestive Tumors, Institut National de la Santéet de la Recherche Médicale, Laboratory of Immunology, Faculté de Médecine, 21033

Dijon, France

ABSTRACT

Peritoneal macrophages from BD IX rats collected 24 hr afteran i.p. injection of Adriamycin (10 mg/kg) were cytotoxic tosyngeneic cancer cells in culture. In contrast, incubation invitro in Adriamycin solutions did not evoke tumoricidal activityin peritoneal macrophages, whatever the incubation time (from1 to 24 hr) and the Adriamycin concentration (from 1 ng to 100/ig/ml). Macrophages incubated with Adriamycin in vitro accumulated the drug in their nuclei, whereas macrophages fromanimals receiving Adriamycin in vivo accumulated it in cyto-

plasmic vacuoles. Early observation of peritoneal cells after invivo exposure to Adriamycin shows that Adriamycin is concentrated in mast cell granules which are released and thenphagocytosed by peritoneal macrophages. Mast cells exposedto Adriamycin in vitro can induce macrophages to becomecytotoxic. These facts explain the difference between macrophages exposed to Adriamycin in vivo and in vitro. Adriamycinfluorescence appears in nuclei of cancer cells incubated within w'vo-labeled macrophages, suggesting that macrophages

can directly transfer the drug into cancer cells and thereforeplay a role in the Adriamycin antitumor effect.

INTRODUCTION

Adriamycin and cyclophosphamide, administered to mice,modify peritoneal macrophages which become cytotoxic tocancer cells in vitro (19). Several mechanisms could explainthis effect. These drugs could directly activate macrophages,they could elicit a release of macrophage-activating factor(s)

by lymphocytes or other cells, or they could be accumulated inmacrophages and transferred into the cancer cells. A comparison between macrophages exposed to these cytotoxic drugsin vitro and in vivo could help to distinguish between thesepossible mechanisms. In this work, we have chosen Adriamycinas the cytotoxic drug since its cellular localization can bedetermined by fluorescence microscopy.

MATERIALS AND METHODS

Animals, Macrophages, and Cancer Cells. All the animals used inthis investigation were syngeneic BD IX rats bred in our laboratory.Resident peritoneal cells were collected after washing peritoneal cavities with 10 or 20 ml of Ham's F-10 medium (Microbiological Associ

ates, Inc., Walkersville, Md.) supplemented with 0.05 mg preservative-free heparin per ml. BCG2-elicited peritoneal cells were collected in

the same way from animals 5 days after the last of 2 i.p. injections offresh BCG (Immuno-BCG F; Institut Pasteur, Paris, France), 1 mg/

injection, 1 month apart. The macrophage content of peritoneal washing fluids was determined either by total leukocyte count and determi-

1Supported by grants from the French League against Cancer and Centre

National de la Recherche Scientifique.2 The abbreviation used is: BCG, Bacillus Calmette-Guérin.

Received July 29, 1981; accepted June 9, 1982.

nation of the percentage of macrophage on Giemsa-stained smears or

by counting cells adhering to a hemocytometer after 30 min of incubation at 37° in a humidified atmosphere. Between 92 and 96% of

adherent cells were characterized as macrophages by their capacityto ingest colloidal carbon particles.

A permanent cancer cell line DHD-K12 was established from a

transplantable colon carcinoma chemically induced in the syngeneicBD IX rat (11). DHD-K12 cells were cultivated in Ham's F-10 medium

supplemented with 4% fetal bovine serum and 8% newborn calf serum.Subcultures between passages 25 and 50 were used for the experiments.

Adriamycin Labeling of the Macrophages. Adriamycin (doxorubi-

cin) was obtained from Sigma Chemical Co. (St. Louis, Mo.) or RogerBellon Laboratories (Neuilly, France). A stock solution, 2.5 mg/ml, wasprepared in 0.9% NaCI solution and stored deep frozen at -20° if not

immediately used. This solution contained no endotoxin on the Limuluspolyphemus amebocyte lysate assay. Peritoneal cells labeled in vivowere obtained by washing peritoneal cavities 1 hr, 24 hr, or 5 daysafter an i.p. injection of Adriamycin, 10 mg/kg body weight, diluted at1 mg/ml in 0.9% NaCI solution. In other experiments, the same amountof Adriamycin was injected in the femoral vein 24 hr before peritonealcell collection. For in vitro labeling, macrophages were incubated for1 to 24 hr in Adriamycin diluted at concentrations between 1 ng and100 /ng/ml in Ham's F-10 medium supplemented with 10% fetal bovine

serum. Macrophages were separated from other peritoneal cells byadhesion to plastic or glass surfaces. In some experiments, Adriamycin-

DNA complex, prepared according to Trouet ef al. (21), was used inthe place of Adriamycin for in vitro labeling.

Adriamycin Localization in Macrophages and Cancer Cells. Adriamycin cellular localization was studied by ultraviolet illumination whichinduced an orange fluorescence at the sites of Adriamycin accumulation. Cells were examined with a Dialux 20 fluorescence microscope(Leitz, Weimar, Germany) equipped with a high-pressure mercury lampand an N-2 filter set. As fixing cells removed Adriamycin or modified its

localization, only living cells were used for this purpose. Peritoneal orcancer cells, cultivated on a glass coverslip, were examined on fluorescence microscopy in a drop of culture medium after turning thecoverslip upside down. Microphotographs were taken on Kodak Tri-X

pan film (400 ASA) with an exposure time of 1 min.Incubation of Macrophages with Adriamycin-treated Lympho

cytes or Their Supernatants. Lymphocytes were prepared from BD IXrat spleens, which were minced with scissors and then passed througha stainless steel mesh. After centrifugation, erythrocytes contaminatingthe pellet were lysed by ammonium chloride (8.5 g/liter) for 1 min.Nucleated cells were carefully washed, counted, and suspended at aconcentration of 107/ml in Roswell Park Memorial Institute Tissue

Culture Medium 1640 (Microbiological Associates, Inc.) supplementedwith 5% endotoxin-free fetal bovine serum. Adriamycin was then added

to reach a final concentration of 0, 0.1, 1, 10, or 100 /¿g/ml oflymphocyte suspension.

In one experiment, lymphocytes were incubated in Adriamycin-con-

taining medium for 4 hr, collected by centrifugation, washed twice,suspended in fresh Roswell Park Memorial Institute Tissue CultureMedium 1640 with 5% fetal bovine serum but without Adriamycin at aconcentration of 107 lymphocytes/ml, and incubated for 40 hr. The

supernatants were then collected by centrifugation, and 100 ¡i\wereadded for 8 hr to wells of Microtest 3040 tissue culture plates (FalconPlastics, Oxnard, Calif.) containing 10s macrophages. Then, supernatants were aspirated and replaced by 10" DHD-K12 cells in 200 p\

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Ham's F-10 medium with 10% endotoxin-free fetal bovine sérum.

Measurement of macrophage-mediated cytotoxicity was performed

after 72 hr.In another experiment, splenic lymphocytes were incubated for 6 hr

in Adriamycin solution, washed 3 times, and then added to a monolayerof 105 peritoneal macrophages in the wells of Microtest 3040 culture

plates, with a ratio of 10 Adriamycin-incubated splenic cells/macro-

phage. After 9 hr of incubation, lymphocytes were washed out andreplaced by 10" DHD-K12 cells. Tumoricidal activity of macrophages

was measured after 72 hr of incubation.Incubation of Macrophages with Peritoneal Mast Cells Exposed

to Adriamycin. Mast cells were isolated from peritoneal cavities ofuntreated BD IX rats by centrifugation on density gradients of Percoli(Pharmacia, Uppsala, Sweden), according to the method described byEnerbäck and Svensson (3). A suspension containing 92% mast cells,4% RBC, and 4% lymphocytes was obtained and incubated for 30 minat 37° in medium supplemented with 10% fetal bovine serum, with or

without Adriamycin, 500/ig/ml. Treated and untreated mast cells werewashed 3 times and then added to a monolayer of 105 peritoneal

macrophages in the wells of microculture plates, with a ratio of 0.15mast cell/macrophage. The same number of mast cells was alsoseeded in control wells, without macrophages. After 16 hr of incubation, control and macrophage-containing wells were washed twice andthen seeded with 10" DHD-K1 2 cells. Tumoricidal activities of macro

phages exposed to medium alone, untreated mast cells, or Adriamycin-

incubated mast cells were compared after 72 hr of incubation. Macrophages exposed to Adriamycin-incubated mast cells were also ob

served on fluorescence microscopy.Cytotoxicity Determination. The cytotoxic effect on cancer cells of

macrophages from normal or BCG-treated animals, with or without

exposure to Adriamycin in vivo or in vitro, was assayed by a quantitativemodification (12) of the assay described by Weinberg et al. (22).Briefly, a volume of peritoneal cell suspension containing 1 x 105

macrophages was transferred to wells of Microtest 3040 tissue cultureplates. After incubation at 37°for 1 hr, each well was washed 3 timeswith Ham's F-10 medium to remove nonadherent cells. In some exper

iments, wells containing macrophages and empty wells used as controls were filled with Adriamycin dissolved in culture medium at variousconcentrations, incubation was continued for a fixed time, and thewells were then washed 3 times with Ham's F-10 medium. Tumor cellswere suspended in Ham's F-10 medium supplemented with 10% fetal

bovine serum (aseptically collected; Microbiological Associates, Inc.),found negative for endotoxin contamination on the L. polyphemusamebocyte lysate assay. Then, 10" tumor cells in 0.2 ml culture medium

were seeded into wells with or without macrophages. After 72 hr ofincubation at 37°,the plates were washed, fixed with 5% formalin, and

stained in a solution of méthylèneblue, 1% in 0.01 M borate buffer, pH8.5. After rinsing, the dye bound to residual cells was eluted with 0.1N hydrochloric acid, and its absorbance was measured on an automaticphotometer (Multiskan; Flow Laboratories, Irvine, Scotland) equippedwith a 630-nm filter. Absorbance of the eluted dye was found to be

proportional to the number of residual target cells. The macrophagesdid not significantly interfere in the assay (12, 22).

The culture conditions (macrophages, cancer cells) were identical inthe 8 wells of the same row. The data obtained from mixed macro-phage-tumor cell cultures were compared to the data obtained fromtumor cells cultivated alone in adjacent wells, using Student's f test for

paired samples. The inhibition produced by macrophages was calculated by the formula:

inhibitiond1\1-- X100

RESULTS

Modification of Peritoneal Cell Count by Adriamycin Injection. When peritoneal cells were collected 1 hr after an i.p.injection of Adriamycin, there was no quantitative change intheir number or their distribution. However, 74% of the macrophages contained basophilic cytoplasmic granules whichstained metachromatically with toluidine blue. These granuleswere found in only 8% of the macrophages from untreatedanimals. About 60% of the mast cells were totally or partiallydegranulated. Many of them were surrounded by several granule-containing macrophages (Fig. 1).

When peritoneal cells were collected 24 hr after i.p. Adriamycin injection, the ratio of leukocyte species was modified(Table 1). The most striking changes were the disappearanceof mast cells and the decrease of eosinophils and lymphocytes.The number of macrophages was not significantly changedand 61 % of these macrophages contained basophilic, meta-

chromatic granules (Fig. 2). Moreover, peroxidase activity wasfound in 72% of these macrophages against 12% in controlanimals. Administration i.v. of Adriamycin did not significantlychange the number, ratio, or morphology of peritoneal cellscollected after 24 hr.

When peritoneal cells were collected 5 days after i.p. Adriamycin injection, there was a significant decrease in the totalnumber of peritoneal cells, including macrophages; 14% of themacrophages contained basophilic metachromatic granules.Mast cells were totally absent. The number of eosinophils andlymphocytes was partially restored (Table 1).

Adriamycin Localization in Macrophages and PeritonealCells. When whole peritoneal cells were examined 1 hr afteri.p. Adriamycin injection, a nuclear fluorescence was observedin all the cells, including macrophages. Fluorescent cytoplasmic inclusions were observed in 65% of the macrophages. Butthe drug was chiefly concentrated in mast cell granules in largeenough amounts to give a pink color when the cells wereexamined by white-light microscopy (Fig. 3).

When macrophages were collected 24 hr after i.p. injectionof Adriamycin, 37 ±4% (S.E.) of them contained fluorescentcytoplasmic inclusions of varied size (0.5 to 5 /im) and color(from dark brown to bright red) (Fig. 4). Nuclear fluorescencewas observed in less than 1% of these macrophages. Thepercentage of Adriamycin-labeled macrophages was higher(75 ±8%) if they were collected from BCG-treated rats. No

Table 1Changes in peritoneal cell composition 1 and 5 days after i.p. injection of

Adriamycin (10 mg/kg)

No. of recovered cells (in millions)

where d1 represents the mean absorbance of wells containing macrophages and cancer cells and d2 is the mean absorbance of wellscontaining target cells alone. Cytotoxicity of macrophages treated ornot treated by Adriamycin was compared by the nonparametric Mann-Whitney U test.

AdriamycinCellsMacrophages

NeutrophilsEosinophilsLymphocytesMastocytesTotal

cellsNo

treatment5.46±1.12a

0.35 ±0.111.69 ±0.370.76 ±0.120.69 ±0.178.95

±1.611

day4.01±0.696

0.46 ±0.1 1b0.11 ±0.03e0.15 ±0.03C0.01 ±0.01d4.74

±0.71e5

days1.82

±0.460.46 ±0.20o0.49 ±0.16d0.31 ±0.07e

0.003.04

±0.74d

Mean ±S.E. of number of cells collected by washing out peritoneal cavities.There were 9 rats in each group.

Not significant.cp< 0.001.d p < 0.01."p<0.05.

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fluorescence was observed in macrophages collected 24 hrafter an i.v. injection of Adriamycin.

When macrophages were examined 5 days after an ¡.p.injection of Adriamycin, cytoplasmic fluorescence was foundin 16 ±4% of the macrophages.

When macrophages were exposed to Adriamycin in vitro atconcentrations of 1 to 100 ng/ml, no fluorescence, cytoplasmic or nuclear, could be detected. When macrophages weretreated in vitro at Adriamycin concentrations of 1 to 100 jug/mlfrom periods of 1 to 24 hr, they accumulated drug in theirnuclei (Fig. 5). Unlike macrophages incubated in vivo, fluorescent cytoplasmic inclusions were observed only in a few macrophages incubated in vitro and were always associated witha brighter nuclear fluorescence. Furthermore, unlike macrophages incubated in vivo, macrophages exposed in vitro for 3to 24 hr at concentrations of Adriamycin higher than 5 /¿g/mlwere morphologically damaged (cytoplasmic blebs) and adhered poorly to glass surfaces.

When splenic lymphocytes or purified peritoneal mast cellswere incubated in Adriamycin in vitro, they accumulated thedrug rapidly, the former in the nucleus, the latter in cytoplasmicgranules. Macrophages incubated with Adriamycin-labeled

lymphocytes did not exhibit cytoplasmic fluorescence. On theother hand, fluorescent cytoplasmic inclusions were observedin 20% of macrophages incubated overnight with Adriamycin-

loaded mast cells (ratio of mast cells to macrophages, 0.15).Adriamycin Transfer from Macrophages to Cancer Cells.

When subconfluent cultures of cancer cells on glass coverslipswere incubated for 48 hr with washed macrophages previouslylabeled with Adriamycin in vivo (10 mg/kg, ¡.p.,for 24 hr) andthen observed with fluorescence microscopy, a distinct nuclearfluorescence was observed in most of the cancer cells (Fig. 6).All the attempts to transfer Adriamycin from macrophagesexposed to Adriamycin in vitro to cancer cells were unsuccessful.

Cytotoxicity Mediated by Adriamycin-labeled Macrophages. Macrophages collected from animals 24 hr after ani.p. injection of Adriamycin were cytotoxic to cancer cells inculture. The cytotoxicity was variable from one experiment toanother but was always significantly superior to the cytotoxiceffect of nontreated macrophages (Table 2). When Adriamycinhad been injected i.v., peritoneal macrophages were not cytotoxic. Macrophages collected 5 days after i.p. administrationof the drug were still highly cytotoxic (66 ±3%). On the otherhand, exposure of macrophages to Adriamycin in vitro did notelicit any cytotoxic effect to cancer cells, whatever the incu-

Adriamycin and Macrophage Tumoricidal Activity

bation time (from 1 to 24 hr) and the Adriamycin concentration(from 1 ng to 100 /¿g/ml). In contrast, the cytotoxic effect ofBCG-elicited macrophages was reduced from 72 to 9% (p <0.001 ) when macrophages had been preincubated in vitro for18 hr in Adriamycin solution (10 jug/ml). Exposure of macrophages from untreated animals for 24 hr to Adriamycin-DNA

complex (Adriamycin concentration, 40 jug/ml), for 9 hr tospleen lymphocytes exposed previously in vitro to Adriamycin(0.1 to 100 jug/ml for 6 hr), or for 8 hr to supernatants ofAdriamycin-pulsed lymphocytes did not induce macrophage-

mediated cytotoxicity to cancer cells. Incubation of normalmacrophages overnight with purified peritoneal mast cellspreincubated 30 min in Adriamycin (500 /xg/ml) induced astrong cytotoxic effect, even if the ratio of mast cell to macrophage was only 0.15. A weaker but significant cytotoxicity waselicited when macrophages were incubated with untreatedmast cells or when cancer cells were exposed to Adriamycin-

labeled mast cells in the absence of macrophages (Table 3).

DISCUSSION

Adriamycin is able to induce a cytotoxic activity in rat peritoneal macrophages when it is administered i.p. This had been

Table 2Effect of i.p. injection of Adriamycin (10 mg/kg) on peritoneal macrophage

toxicity to cancer cellsAbsorbance of cancercells"Experiment1234Without

macrophages901e316413851With

normal macrophages926

(+3)d

935 (+4)928(+3)287

(-9)300(-5)396

(-4)396(-4)848

(0)714 (-16)With

macrophagesfrom Adriamycin-

treatedrats6744

(-17)8701 (-22)615(-32)78

(-75)106(-66)116

(-72)85(-79)299

(-65)170 (-80)

Absorbance is measured in arbitrary units." Macrophages collected 24 hr after an i.p. injection of Adriamycin, 10 mg/

kg.cMean of absorbances measured in 8 microculture wells.Numbers in parentheses, percentage oígrowth enhancement ( + ) or cyto

toxicity (-) determined by comparison to cancer cells cultivated without macro

phages.8 In each experiment, cytotoxic effect induced by macrophages was signifi

cantly superior after Adriamycin treatment.

Table 3Effect of incubation with Adriamycin-loaded mast cells on peritoneal macrophage toxicity to cancer cells

MastcellsNo.0015.10315.10315.10315.103TreatmentNoneNoneAdriamycin9Adriamycinphagesa010=010s010scancer

cells425+ 17C409

±35451±21347±49354±35211±34cytotoxicity4%

(NS0)23%

(p<0.01)40%

(p< 0.001)

Macrophages were incubated overnight with or without mast cells and then washed before addition ofcancer cells.

b Absorbance is measured in arbitrary units.c Mean ±S.D. for 8 wells.d NS, not significant.e Mast cells were incubated for 30 min in Adriamycin. 500 /ig/ml.

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demonstrated previously in mice by Stoychkov et al. (19),Santoni et al. (17), and Haskill (5). However, we did not find acytotoxic activity in peritoneal macrophages when the drugwas injected i.V.; the same observation has been reported byMantovani (9).

The tumoricidal effect of macrophages exposed to Adria-

mycin in vivo could be explained by several mechanisms. First,Adriamycin could directly activate macrophages. A direct activation of macrophages to cytotoxicity has been observed withother agents, for instance bacterial endotoxins (1). However,we have been unable to induce a tumoricidal activity in peritoneal macrophages exposed in vitro to a large range of Adriamycin concentrations. Furthermore, Adriamycin exposure invitro inhibits destruction of tumor cells by activated macrophages from BCG-treated rats.

Another mechanism could be an indirect activation of macrophages by Adriamycin through the release of macrophage-

activating factor, a lymphokine, by peritoneal lymphocytes.However, we have shown that a supernatant of lymphocytespulsed for 4 hr in a large range of Adriamycin concentrationsdid not evoke any cytotoxic activity in normal peritoneal macrophages.

A third explanation of the toxic effect on tumor cells couldbe a transfer of Adriamycin from macrophages to tumor cells.This hypothesis is supported by fluorescence examination.Macrophages from Adriamycin-treated animals are loaded withcytoplasmic Adriamycin-containing inclusions. The same ob

servation was made by Haskill (5). After incubation with thesemacrophages, cancer cells are labeled and the Adriamycinfluorescence is located in their nuclei.

The mechanism of the transfer of Adriamycin from the macrophage to the cancer cell is not yet established. A simplerelease of Adriamycin from macrophage to the culture mediumis improbable. The concentration of Adriamycin in the mediumwould be too low to kill DHD-K12 cells, which are able to resistAdriamycin concentrations up to 1 /ig/ml for 72 hr.3 Further

more, Stoychkov et al. (19) and Haskill (5) demonstrated thatthe supernatant of Adriamycin-treated macrophages is not

toxic to cancer cells. Passage of Adriamycin into the cancercell could be the result of a lysosomal transfer which has beensuggested previously by Hibbs (6) and Buccana ef al. (2) forother lysosomal labels. Finally, Adriamycin-loaded particles

could be actively ingested by cancer cells with a part of themacrophage cytoplasm, as we have observed on phase-contrast cinemicrography with India ink-labeled macrophages (13).

This could be supported by the observation of Haskill (5) thatdisrupted Adriamycin-elicited macrophages were still cytotoxic

to cancer cells.It is important to explain why Adriamycin localization was

different in macrophages treated by this drug in vitro and invivo. Macrophages exposed to Adriamycin in vitro accumulatethe drug in their nuclei, as it has been previously reported (4).This localization results in the alteration of macrophage morphological and functional properties, including tumoricidal activity. On the other hand, macrophages from Adriamycin-

treated animals accumulate the drug in their cytoplasm and aretoxic to cancer cells. At first, we supposed that macrophagescould endocytose the drug not as a free form but as Adriamy-cin-nucleic acid complex or that they could ingest Adriamycin-

' Unpublished results.

labeled nuclei from other peritoneal cells such as lymphocytes.However, we were unable to label the cytoplasm of macrophages or activate macrophage cytotoxicity by incubation withAdriamycin-DNA complex or Adriamycin-labeled lymphocytes.

The probable answer comes from the examination of peritoneal exúdate cells collected only 1 hr after i.p. Adriamycininjection. All the cells had a nuclear fluorescence, but therewas chiefly a strong accumulation of Adriamycin in mast cellgranules, sufficient to stain them pink with white-light micros

copy. It was also observed that macrophages phagocytosedAdriamycin-labeled granules from degranulating mast cells.Twenty-four hr after i.p. Adriamycin injection, mast cells haddisappeared and a majority of macrophages contained baso-

philic, metachromatic inclusions very similar to the mast cellgranules. It may be supposed that Adriamycin is rapidly concentrated in mast cell granules which are then released byAdriamycin-induced mast cell degranulation and ingested bymacrophages. Endocytosis of mastocytoma basophilic granules by peritoneal macrophages has been reported previously(8). Granule-containing macrophages found 24 hr after Adriamycin injection are probably not resident, peroxidase-negative

macrophages which have been destroyed by exposure to highdrug concentration. They are probably inflammatory, peroxi-dase-positive macrophages, newly recruited from blood andbone marrow following Adriamycin-induced peritoneal inflam

mation. As the Adriamycin concentration rapidly decreases inthe peritoneal cavities (7), these macrophages are not damaged and their nuclei are not labeled.

The role of macrophages in tumoricidal properties of Adriamycin in vivo is a point which has to be clarified. If macrophagesare really able to concentrate Adriamycin and transfer it to thecancer cell, they could act as a Trojan horse and play a role inthe clinical efficiency of Adriamycin or other cytotoxic drugs.Adriamycin is not the only cytotoxic agent able to induce atumoricidal activity in peritoneal macrophages. The same effecthas been reported with cyclophosphamide (19) and mitomycinC (16). A role of macrophages in the tumor response toAdriamycin in vivo is supported by the results obtained byTagliabue ef al. (20) and Mantovani ef al. (5). They found thatthe antitumoral effect of Adriamycin on several experimentaltumors was enhanced by macrophage-stimulating agents(BCG, Corynebactehum parvum) and inhibited by macrophage-

damaging agents (silica, carrageenan). The hypothesis of arole of the macrophage in the transport of cytotoxic drugs tothe tumor cell could also explain the favorable results of thecombined chemotherapy and immunotherapy in several experimental tumor models (10, 14, 15, 18).

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2. Bucana. C., Hoyer, L. C., Hobbs, B., Breesman, S., McDaniel, M., andHanna, M. G. Morphological evidence for the translocation of lysosomalorganelles from cytotoxic macrophages into the cytoplasm of tumor targetcells. Cancer Res., 36. 4444-4458. 1976.

3. Ennerbäck, L., and Svensson, I. Isolation of rat peritoneal mast cells bycentrifugaron on density gradient of Percoli. J. Immunol. Methods. 39:135-145, 1980.

4. Facchinetti, T., Raz. A., and Goldman, R. A differential interaction of dau-nomycin, Adriamycin and N-trifluoroacetyl-adriamycin-14-valerate withmouse peritoneal macrophages. Cancer Res., 38: 3944-3949, 1978.

5. Haskill, J. S. Adriamycin-activated macrophages as tumor growth inhibitors.Cancer Res., 41: 3852-3856. 1981.

6. Mibbs, J. B. Heterocytolysis by macrophages activated by Bacillus Calmette-Guerin: lysosome exocytosis into tumor cells. Science (Wash. D. C.), 184:

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7. Johansen, P. B. Doxorubicin pharmacokinetics after intravenous and intra-peritoneal administration in the nude mice. Cancer Chemother. Pharmacol.,5. 267-270, 1981.

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11. Martin, F., Knobel, S., Martin, M. S.. and Bordes, M. A. carcinofetal antigenlocated on the membrane of cells from rat intestinal carcinoma in culture.Cancer Res., 35. 333-336. 1975.

12. Martin. F., Martin, M. S., Jeannin, J. F., and Lagneau, A. Rat macrophage-mediated toxicity to cancer cells: effect of endotoxins and endotoxin-inhib-itors contained in culture media. Eur. J. Immunol., 8. 607-611, 1978.

13. Martin, F.. Olsson. N. O , Jeannin, J. F., and Martin, M. S. Tumor cell killingby activated macrophages: role of the endocytosis of macrophage fragmentsby cancer cells. In: L. Israel. P. Lagrange, and J. C. Salomon (eds.). CancerImmunology and Parasite Immunology, pp. 279-286. Paris: Institut National

de la Santéet de la Recherche Médicale, 1980.14. Mihich. E. Combined effects of chemotherapy and immunity against leukemia

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Adriamycin and Macrophage Tumoricidal Activity

15. Moore, M., and Williams, D. E. Contribution of host immunity to cyclophos-phamide therapy of a chemically-induced murine sarcoma. Int. J. Cancer,11: 358-368, 1973.

16. Ogura, T.. Shindo, H., Namba, M., and Yamamura. Y. Tumoricidal activity ofperitoneal exúdate cells from rats treated with mitomycin C. Gann, 71.920-921. 1980.

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21. Trouet. A., Deprez de Campeneere, D., de Smedt-Malengreaux, M., andAtassi, G. Experimental leukemia chemotherapy with a lysosomotropic Ad-riamycin-DNA complex. Eur. J. Cancer, 10: 405-410, 1974.

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Fig. 1. Peritoneal cells collected 1 hr after an i.p. injection of Adriamycin (10 mg/kg). A degranulating mast cell is surrounded by 3 macrophages containingbasophilic granules. Glemsa, x 650.

Fig. 2. Peritoneal cells collected 24 hr after an i.p. injection of Adriamycin. Basophilic granules in the cytoplasm of 7 macrophages. Giemsa, x 1250.Fig. 3. A mast cell, collected 1 hr after an i.p. injection of Adriamycin, stains red-orange with fluorescence microscopy (left) and pink with white-light microscopy

(right), x 450.

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Adhamycin and Macrophage Tumohcidal Activity

Fig. 4. Peritoneal macrophage collected 24 hr after an i.p. injection of Adriamycin. Adriamycin is collected in cytoplasmic vacuoles, x 2500.

Fig. 5. A peritoneal macrophage incubated in vitro for 6 hr in Adriamycin, 20 /ig/ml. Fluorescence is localized to the nucleus, x 2500.Fig. 6. Culture of DHD-K12 cancer cells incubated for 48 hr with peritoneal macrophages from Adriamycin-treated rat. Cancer cell nuclei are stained by

Adriamycin transferred from in v/Vo-labeled macrophages. Fluorescence, x 450.

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1982;42:3851-3857. Cancer Res   François Martin, Anne Caignard, Olivier Olsson, et al.  

in Vitro or VivoinTumoricidal Effect of Macrophages Exposed to Adriamycin

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