intraperitoneal administration of corynebacterium parvum in patients with ascitic ovarian tumors...

Int. J . Cancer: 27,437-446 (1981)

INTRAPERITONEAL ADMINISTRATION OF CORYNEBACTERIUM PARVUM IN PATIENTS WITH ASCITIC OVARIAN TUMORS RESISTANT TO CHEMOTHERAPY: EFFECTS ON CYTOTOXICITY OF TUMOR-ASSOCIATED MACROPHAGES AND NK CELLS

Alberto MANTOVANI’, Cristiana SESSA~, Giuseppe PERI’, Paola ALLAVENA’, Martino INTRONA’, Nadia POLENTARUTTI and Costantino MANCIONI~

Ginecologica, University of Milan, Via Commenda, 12 - 20122 Milan, Italy. Istituto di Ricerche Farmacologiche “Mario Negri”, Via eritrea, 62 - 20157 Milan; and 21 a Clinica Ostetrica e

Eight ovarian cancer patienk with ascites resistant to various chemotherapy protocols were treated i.p. with C.parvum in an effort to obtain a palliative reduction of peritoneal effusions. C.parvum (7- I 4 mg) was given i.p. on days 0, 7, 28 and then at monthly intervals. Three patients showed no clinical evidence of therapeutic benefit from C.parvumadministration. Three subjeck had a complete disappearance of ascites, and two additional patients showed a marked reduction of their effusions. Solid tumor masses were not affected by i.p. C.parvum. C.parvum injection caused a rapid decrease of the percentage of tumor cells in the ascites as early as 7-1 5 days after the first treatment. Concomitantly, the percentage of polymorphs and, to a lesser extent, of macrophages in the ascitic tumor increased, whereas tumor- associated lymphoid cells (TAL) were not altered in a consistent pattern. The tumoricidal activity of peripheral blood monocytes and natural killer (NK) cells was not consistently modified after C.parvum treatment. Pre- treatment NK activity of TAL was usually very low and was not appreciably modified by i.p. C.parvum. In one patient, whose TAL had NK activity in the range of normal PBL, C.parvum administration was followed by a profound depression of intratumor NK cytotoxicity. The tumoricidal activity of tumor-associated macrophages (TAM) was not enhanced following C.parvum administration; 14-2 I days after the first C.parvum in- oculation, a marked decrease of the cytolytic capacity of TAM was observed, cytotoxicity returning to pretreatment values thereafter. These observations do not elucidate the mechanism of the limited, but significant, anti-tumor activity of i.p. Cparvum in human ovarian carcinomatous ascites. The lack of enhancement of macrophage cytotoxicity after i.p. C.parvum, predicted on the basis of data in rodents, cautions against the empirical use in humans of agenk assumed to affect human neoplasia through activation of host defense mechanisms.

Corynebacterium parvum (Cparvum) has pro- founds effects on host defense mechanisms in rodents and has significant anti-tumor activity in selected experimental systems (Scott, 1974). Sys- temic administration (i.v. or i.p.) of this agent en- hances murine macrophage-mediated cytotoxicity (Ghaffar et al., 1975; Olivotto and Bomford, 1974) and natural killer activity (Herberman et al., 1977; Oehler et al., 1978; Ojo et al., 1978; Flexman and Shellam, 1980). There is evidence that the antitumor activity of i.v. and i.p. C.parvum is related, at least in part, to stimulation of macrophage and/or NK cell function (Scott, 1974; Woodruff and Warner, 1977).

In several studies Cparvum has been given S.C. or i.v. to tumor-bearing patients, but it has not been ascertained whether activation of macrophages and

NK cells, which according to animal data play a role in the anti-tumor efficacy of this agent, also occurs in humans. In one study, i.p. injection of C.parvum was found to reduce ascites in patients with ovarian tumors resistant to chemotherapy (Webb et al., 1978). Ovarian carcinomatous ascites provides an easily accessible source of tumor cells, macrophages and lymphoid cells in suspension, and in previous studies we have investigated the tumoricidal activity of macrophages and NK cells isolated from these tumors (Mantovani et al., 19796; 1980a, d ) . The present study was designed to evaluate the efficacy of i.p. C.parvum for the palliative treatment of ovarian ascitic tumors resistant to chemotherapy and the effects of this agent on tumor-associated macrophages and NK cells in humans.

PATIENTS AND TREATMENT

Eight patients having ovarian tumors with ascites resistant to first and second line chemotherapy entered this study. Six had a serous adenocarcinoma, one had an endometrioid adenocarcinoma, and one a granulosa cell tumor. Drugs which had been employed for the treatment of the serous and endometrioid carcinomas included alkylating agents, ad- riamycin (ADM), hexamethylmelamine (Hexa), cis platinum (11) diamine dichloride (DDP), methotre- xate (MTX), 5-fluorouracil (5FU) and medroxy- progesterone acetate (MPA). The patient with a granulosa cell tumor had been treated with vincris- tine (VCR), actinomycin D (ActD) and cyclophos- phamide (CTX). These patients had no chance of benefit from further treatment with cytotoxic agents. Patients admitted to the study had been submitted to repeated paracenteses during the previous 4 weeks, with progressive increase in ascitic fluid production.

C.parvum (Coparvax, Wellcome Research Laboratories, Beckenham, England) was adminis- tered i.p. at a dose of 14 mg in 200 ml saline during a 1-h infusion. This treatment was repeated 7 days later and, if there was evidence of reduction of ascites a 7 mg dose was injected on day 28 after the first administration. The original protocol for patients showing evidence of reduction of ascites planned a subsequent monthly administration of 7 mg C.parvum, but two subjects whose effusions completely disappeared refused further treatment because of

Received: December 5 , 1980 and in revised form Febru- ary 2, 1981.

438 MANTOVANI ET A1

the side-effects. Before infusion of Cparvum, samples of ascites (usually 500-1,000 ml) and of peripheral blood (10-20 ml) were obtained for in vitro assays.

Following i.p. injection of Cparvurn, clinical findings were essentially as previously described (Webb et al., 1978) and only the essential features will be described here. Toxicity included: gastrointestinal pain (8/8 patients); nausea and vomiting (718); fever (5/8 patients, up to 393°C for up to 24 h). After the second Cparvum treatment, one patient showed se- vere dyspnea and hypotension, which required treatment with 100 mg hydrocortisone; treatment was discontinued thereafter.

As observed after i.v. injection of Cparvum (Gill et al., 1980), blood polymorphs were markedly increased 24 h after i.p. Cparvum, and returned to normal values thereafter. For instance, pretreatment polymorphs on day 0 were 4.435 k 0.942 X 10'/ml (mean k S D , range 2.948-5.460) and were 9.153 k3.02 (range 5.475-13.521) 24 h after Cpar- vum administration (p <0.01). No other consistent alteration of hematological values was detected.

Two patients showed a complete disappearance of ascitic effision after two Cparvum adminiastrations and one after the first dose of this agent. They have remained free of effusion for 2, 6 and 13+ months respectively. In two additional subjects, definite evidence of marked reduction of ascites formation was obtained, as assessed by measuring the abdominal diameters at the umbilical level and 8 cm above and below, and by a reduction in the requirement for paracentesis for relieving ascites; however the effusions did not completely disappear. In one of these two subjects loculation of the ascites was suspected. Three patients obtained no appreciable benefit from Cparvum treatment. In none of the subjects was any indication obtained for an effect of C.parvum on solid tumor masses.

MATERIAL AND METHODS

Cellular compositions of ascites Smears of ascites cells were stained with Papa-

nicolao or Giemsa in either of the two cooperating institutions. Codes samples were read at the end of the series of experiments. Two of us, unaware of the code, read the smears independently in a blind fashion and the average of the two determinations was calculated. Two hundred cells were counted. It must be stressed that, in addition to the bias inherent in a purely morphological classification of cell types, ovarian carcinoma cells frequently occur in clumps and evaluation of tumor cell numbers in them is dif- ficult.

Peripheral blood lymphocytes (PBL) Peripheral blood (10-20 ml) was obtained from

ovarian cancer patients or from 97 control subjects. Blood was diluted 1:4 with phosphate-buffered saline (PBS, Eurobio, Paris, France) and 40 ml on 10 ml Ficoll-Hypaque (Eurobio) were centrifuged at 400 g for 20 min at room temperature. Mononuclear cells were collected at the interface and washed with PBS, then 10-30X10' cells were incubated in 10-

20ml of RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS, GIBCO-Biocult, Glasgow, Scotland) for 45 min at 37°C in plastic Petri dishes (3003, Falcon, Oxnard, CA). Non-adherent cells were collected, centrifuged at 400 g for 5 min and finally resuspended in RPMI 1640 medium with 10 % FBS and 50 &ml gentamycin. Non-adherent cell preparations were only partially depleted of monocytes as 2-4 % of these cells were mononuclear phagocytes, as assessed by morphology, avid uptake of neutral red and staining for non-specific esterase as compared to 10-30 % of the original mononuclear cell suspensions (Mantovani et al., 1980d). Tumor-associated lymphocytes (TAL)

Enriched preparations of TAL were obtained by stepwise application of density and velocity sedimen- tation on discontinuous Ficoll-Hypaque or FBS gradients (Vose et al., 1977a, b; Mantovani et al., 1980~). Carcinomatous ascites was centrifuged at 400 g for 5 min. Cells were resuspended in PBS and 20 ml of the cell suspension (1-5 X 10' cells/ml) were stratified on 10 ml Ficoll-Hypaque. The tubes (25330, Corning, NY) were then centrifuged at 400 g for 20 min at room temperature and the mononuclear cell layer was aspirated. To remove macrophages from ovarian ascites cell suspensions, mononuclear cells in serum-free medium (Manto- vani et al., 1979b, 1980d) were incubated for 45 min at 37°C in plastic Petri dishes. The procedure was repeated 2-3 times until contamination with mononuclear phagocytes, assessed by morphology and avid uptake of neutral red, was less than 5 %. After washing with 50 ml PBS, 10 ml of the mononuclear cell suspension (-2 X 106/ml) were layered on discontinuous gradients of 10 ml of 100% and 15 ml of 75 % Ficoll-Hypaque. The tubes were centrifuged at 1700 rpm for 10 rnin at room temperature. Lympho- cyte-enriched suspensions were harvested at the 75 %-lo0 % interface and tumor cells were found on top of the 75% fraction. The procedure was repeated once or twice if separation was not satisfactory. When separation of lymphocytes from tumor cells on discontinuous Ficoll-Hypaque gradients was not satisfactory, as judged by morphological examination of Wright-stained smears, cell suspensions (10- 20 X 106/10 ml PBS) were layered onto 10 ml FBS and incubated at room temperature for 60-90 min; tumor cells sedimented at the bottom of the discontinuous gradient whereas cells with lymphoid morphology remained in the upper part of the gradient. The various phases of the separation procedure were monitored by morphological examination of Wright- stained smears. Cell suspensions from the unsepa- rated carcinomatous ascites and occasionally from the purified lymphocytes or tumor cells were stained with Papanicolao and examined by independent pathologists. Lymphoid cell suspensions were ac- cepted for use when at least 95 % (usually more than 99%) of the cells had the typical unequivocal morphology of the respective cell type. Less than 5 5% of the lymphoid cell suspensions belonged to the monocyte-macrophage lineage, as assessed by morphology, rapid uptake of neutral red and non-specific esterase staining. Viability of enriched fractions al- ways exceeded 90 % and lymphocyte recovery

INFLAMMATORY CELLS IN HUMAN TUMORS 439

100-

I I 50- 1

- PI U

0-

ranged from 50 to 0.5 X lo6 cells for different patients. When normal PBL were submitted to the same separation procedure as used for ovarian tumors, NK activity was not significantly affected.

Control peritoneal macrophages Peritoneal exudate macrophages were obtained

from 36 patients undergoing surgery for benign non- malignant gynecological diseases as previously described (Mantovani et al., 1980b, d). Peripheral blood monocytes

Peripheral blood monocytes were obtained by adherence on microexudate-coated plastic and ethy- lene diamine tetraacetic acid (EDTA) as previously described in detail (Mantovani et al., 1979a, 1980d). Briefly, mononuclear cells (10-30 X lo6) were incubated in 10-20 ml of RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS, GIB- CO-Biocult, Glasgow, Scotland, or aseptically collected FBS, Microbiological Associates, Walkersvil- le, Md.) for 45 min at 37°C in plastic Petri dishes (3003, Falcon, Oxnard, Calif.) conditioned as previously described (Mantovani et al., 1980d). After non-adherent cells had been thoroughly washed off, monocytes were recovered by exposure for 5-15 min to 1 mM EDTA in phosphate-buffered saline. After washing with 50 ml of basal medium Eagle (BME), cells were resuspended in RPMI 1640 medium with 10% FBS. Under these conditions, more than 90% of the adherent cells belonged to the monocyte-macrophage lineage as assessed by morphology, staining for non-specific esterase, avid uptake of neutral red, binding and phagocytosis of antibody-coated sheep erythrocytes.

Tumor-associated macrophages (TAM) To isolate macrophages from ascitic tumors, a re-

cently described method (Mantovani et al., 1980d) was employed. Adherence was performed on microexudate-coated plastic, thus allowing recovery of cells with EDTA and accurate assessment of attacker to target (A:T) ratios. Briefly, mononuclear cells obtained by centrifugation on Ficoll-Hypaque were incubated for 45 min at 37°C in BME (1O-20X1O6/ 10ml) on conditioned Petri dishes (3003 Falcon, Ox- nard, CA). At the end of the incubation, non-adherent cells were removed with jets of medium and macrophages were recovered by exposure to EDTA as described for monocytes. As previously reported for peripheral blood monocytes and tumor-associated macrophges (Mantovani et al., 19796, 1980d) more than 90 % of the adherent cells were mononuclear phagocytes as assessed by morphological and functional criteria. Target cells

The SV40-transformed mKSA TU5 kidney line (TU5; Kit et al., 1969) was used as target for monocyte or macrophge-mediated lysis and was main- tained by serial passages in RPMI 1640 medium with 20% FBS. This tumor line was selected to assess cytolytic activity levels since previous studies had shown that it is susceptible to the cytocidal activity of adherent peripheral blood mononuclear cells and circumstantial evidence suggests that effector cells

are cells of the monocyte-macrophage lineage (Man- tovani et al., 1979a). Non-confluent cultures were prelabelled by overnight exposure to 0.5 yCilml of [methyL3H] thymidine (6Ci/mmole, Radiochemical Centre, Amersham, England) in 5 ml growth medium in 25 cm2 tissue culture flasks (Costar, Cam- bridge, MA). At the end of the incubation, cells were detached by exposure to 0.25 % trypsin- 0.02% EDTA for 5 min. After washing twice with 50 ml medium, TU5 cells were finally resuspended in RPMI 1640 medium with 10% FBS.

To measure NK activity, the K562 line (Lozzio and Lozzio, 1973) was used. Cells (1-5 X lo6 in 1 ml growth medium) were incubated with 20-50 ml pCi of W r (Sodium Chromate, Radiochemical Centre, Amersham, England) at 37°C for 45 min. Labelled cells were washed twice with 50 ml of medium before use in NK lysis assays.

Macrophage-mediated cytotoxicity Cytolytic activity of monocytes and macrophages

was evaluated as [3H] thymidine release from prelabelled TU5 cells as previously described (Manto- vani et al., 1979a, 1980d). Briefly, TU5 cells (lo4/ sample) were incubated at 37°C in air with 5 % CO, for 48 in a final volume of 0.3 ml of RPMI 1640 medium with 10% FBS in flat-bottomed 6.4 mm culture wells (3596 Costar, Cambridge, Ma). A range of attacker to target cell (A:T) ratios (5:1, l O : l , 20:l and 40:l) were usually employed. Tumor cell growth was checked daily under an inverted microscope. At the end of the incubation 0.1 ml of the supernatant was collected and counted in a Nuclear Chicago li- quid scintillation spectrometer. To assess total incorporated radioactivity, which for TU5 averaged 6250 cpm, tumor cells were incubated with 1% sodium dodecyl sulfate (SDS) in water. The percentage of isotope release was calculated as 100 X (AlB), where A is the isotope release in the supernatant and B is the SDS-releasable radioactivity. Specific lysis was calculated by subtracting spontaneous release of TU5 cells in the absence of mononuclear phagocytes, which did not exceed 25 %.

A

A

4

f t. A

A.. f

-LA- Tumor cc115 mlymorphs Macrophages Lymphoid cells

FIGURE 1 - Cellular composition of ovarian ascites before treatment with C. purvum. A, patients who subsequently showed no clinical evidence of response to therapy.

440 MANTOVANI ET A1

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In preliminary experiments, two blood samples were obtained from the same laboratory donors and monocytes were separated and tested in parallel. Cy- totoxicity values of preparations from the same donor were superimposable. Moreover, three normal laboratory donors were repeatedly tested (five or six times) for monocyte cytotoxicity over a period of 12 months. The day-to-day coefficient of variation (= SD/tnean) of the assay was 0.26 -+ 0.13 (mean f

N K cytotoxicity assay 51Cr-labelled tumor cells ( 2 X lo4) were cultured

with as many A:T ratios as possible (usually 50:1, 2S:1, 12:l and 6:l) in 0.2 ml growth medium in round-bottomed wells of microplates (Sterlin, Ted- dington, Middlesex, England). The routinely employed incubation time was 20 h, but in a number of experiments lysis was also measured after 4 h incubation. Isotope release wascalculatedas (A/B) X 100, where A is the isotope in the supernatant and B is the total incorporated radioactivity released by incubation with 1 % SDS. Specific lysis was calculated by subtracting spontaneous isotope release of tumor cells alone. Spontaneous 51Cr release from K562 cells was O.S-l.S% per hour of incubation.

In each NK experiment, ovarian cancer lymphocytes were tested along with cells from at least three control subjects. A semilog plot of the specific cytotoxicity data against the number of effector cells

SD) .

per sample was obtained; the number of effector cells required for 33 95 specific lysis was graphically determined and arbitrarily defined as one LU 33. Expression of NK cytotoxicity results relative to normal subjects tested concomitantly in the same experiment has been reported to consistently reduce the day-to-day CV of the assay (Pross and Baines, 1976; Santoli et al., 1978), as also observed with blas- togenesis tests (Dean et al., 1977). This was confirmed in previous experiments in this laboratory (Mantovani et al., 1980~). Therefore, for each lymphoid cell preparation an RCI was calculated as: XnLU33hLU33, where XnLU33 is the average LU33 value for control subjects tested concomitantly and tLU33 is the same value for the test subject. Thus, an RCI of 0.2 indicates that five times more test lymphocytes are required to obtain 33% lysis when compared to control effector cells.

The day-to-day CV of the NK assay, when data were expressed as RCI, was 0.27 f 0.02 for PBL from four normal laboratory donors repeatedly tested over a period of 4 months.

Statistical analysis

multiple range test. Cytotoxicity data were analysed by Duncan's new

RESULTS

Although eight patients entered the study and were given C. parvum, for one of these, who showed a reduction of ascites, but not a complete disappearance, no baseline pretreatment value was obtained. Moreover, for various reasons (e.g. , difficulty in ob- taining sufficient amounts of blood; low cell numbers recovered after fractionation of ascites, etc.) a complete follow-up could not be carried out for each of the subjects evaluated at time 0.

Figures 1-3 show the cellular composition of carcinomatous ascites and the cytotoxic activity of blood and tumor-associated mononuclear phagocytes and N K cells before treatment with C. parvum.

.A.

i.

- - ~

-IoJ mo$tes macrophages monocytes TAM c o n t r o l sublecls ovarian cancer patients

FIGURE 3 - Cytolytic activity of monocytes or macrophages in ovarian ascitic tumors before treatment with C. parvum. The A:T ratio was 20: 1. A, patients who subsequently showed no clinical evidence of response to therapy.

INFLAMMATORY CELLS IN HUMAN TUMORS

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90-

80-

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Day a f t e r the f i r s t C . p a r v u m treatment D a y a f t e r t h e f i rs t C.parvum treatment

FIGURE^ - Effect of i.p. c. parvum on the cellular composition of ovarian carcinomatous ascites. A, patients who were clinically unresponsive to therapy.

The percentages of the various cell types, evaluated on a purely morphological basis, varied considerably among the seven patients from whom a cell smear taken before time 0 could be examined (Fig. 1). Re- sistance or susceptibility to C. parvum treatment was not associated with recognizable patterns of cell composition of the ascites before starting treatment.

In agreement with previous data on NK in ovarian carcinoma, three out of seven subjects had clearly depressed blood NK activity (RCI <0.4). Depres- sion of NK cytotoxicity was more marked in TAL, four of five preparations having RCI values below 0.4 (Fig. 2). The tumoricidal activity of blood monocytes and TAM was similar to control values for blood and peritoneal cavity (Fig. 3). Lack of respon- siveness to C. parvum therapy was not associated with a recognizable pattern of macrophage or NK cell-mediated cytotoxicity in the blood or ascitic tumors of this small series of patiens (Fig. 2 and 3). It should be noted that pretreatment values shown in Fig. 1-3 are representative of various experiments performed before giving C. parvurn because these subjects required frequent paracenteses for pallia- tion (see "Patients and treatment").

In five subjects, smears of unfractionated ascites cells were obtained at different times after starting

C. parvum therapy and examined in a blind fashion (Fig. 4). In all five subjects a decrease in the percentage of tumor cells was detected as early as 7-14 days after the first C. parvum injection (Fig. 4a). In one patient, who was clinically unresponsive to therapy and from whom ascites samples were obtained also at later times, the decrease in the percentage of tumor cells was only transient (Fig. 4a). All patients showed an increased percentage of polymorphs in their effusions in the first 20 days after entering the C. parvum protocol (Fig. 4b) and in two subjects polymorphonuclear leukocytes represented up to 70- 90% of the ascitic cells. In four of five patients a small increase in the percentage of TAM was also observed (Fig. 4c). No increase in the percentage of morphologically identified macrophages occurred in a fifth, clinically unresponsive subject. Alterations in the percentage of morphologically defined lymphoid cells did not follow a consistent, recognizable pattern (Fig. 4 4 .

Macrophages and lymphoid cells were purified from ovarian ascitic tumors and recoveries of these cell types are presented in Figure 5. The number of macrophages rccovered from 1 ml of effusion fluid was markedly increased (up to 20 fold) after C. parvum administration in all subjects for whom these

442 MANTOVANI ET AL

data were obtained (Fig. 52). Conversely, the lymphoid cell recovery was not altered in a consistent way in the four patients for whom these data could be obtained.

Blood NK activity and monocyte cytotoxicity was monitored in six and five patients respectively (Fig. 6a and 8a). Given the day-to-day variability of the assays (CV of 0.26 and 0.27 for monocyte and NK cytotoxicity) and the limited number of subjects examined, no consistent pattern of modification of these reactivities emerged following C. parvurn treatment.

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In four patients, NK activity of T A L was mea- I r I ..--+-I sured at different times after entering the C. parvurn 0 10 2 0 30 40 50 100 200 300 D a y a f t e r t h e f i r s t C parvum treatment

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Z 0-1 I I I /+--

0 10 2 0 30 40 5 0 100 200 300 D a y a f t e r t h e f l r s i C p a r v u m t r e a t m e n t

FIGURE 6 - NK activity of PBL ( a ) and TAL ( 6 ) after i.p. I I I ,- treatment with C. parvum. 0 , Repeatedly tested normal o 1 o 2 b 30 4 o 5; 100 laboratory donors; A ovarian cancer patients clinically unre-

sponsive to C. parvum; 0, clinically responsive ovarian cancer patients.

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0 so

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protocol (Fig. 6b) . In three of these NK activity of T A L was very low (RCI <0.4) and it was not consistently altered following C. parvum administration. Pretreatment NK activity of T A L from a fourth sub- / - ---

-- --A

ject was in the range of normal PBL (RCI = 0.8).

T A L from this subject, who showed a complete,

duced to an RCI of o.l 2 weeks after the first treatment. NK cytotoxicity data for this subject, tested at different A:T ratios, are presented in Figure 7.

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D a y a t t r r the f i r s 1 C p a r " = I r e a t m e n 1

FIGURE 5 - Effect i.p. C. parvum on the recoveries of temporary (2 months) regression Of ascites, was re- lymphoid cells (6) and macrophages ( a ) from ovarian carcinomatous ascites. A, patients who subsequently showed no clinical evidence of response to therapy.

T A L P a t l e n t ' s P E L C o n t r o l PBL 1

- 6 I 1 2 1 2 5 1 5 0 1

A 1 r.110

FIGURE 7 - NK activity of TAL from one patient (No. 1) tested on different days after the first C. parvum treatment. Numbers in paren- thesis indicate the recoveries of lymphoid cells (per ml) from ascites or blood. Cytotoxicity and recovery values for control PBL are means of at least two normal donors.


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FIGURE 9 - Tumoricidal activity of TAM from one patient (No. 5 ) tested on different days after the first C. .",, (15,,01i parvum treatment. Numbers in parentheses indicate the recoveries of mononuclear phagocytes per ml of blood or ascites. Cytotoxicity and re-

*."11155.10'I * ~ . y 0 i v 5 . 1 0 ' i covery values for control monocytes, tested in parallel, are means of at least two normal donors, except for

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FIGURE 8 - Tumoricidal activity of blood monocytes (a ) and TAM ( b ) after i.p. treatment with C. parvum. The A:T ratio was 20:l. 0 , Repeatedly tested normal laboratory donors; A, ovarian cancer patients clinically unresponsive to C. parvum; 0, clinically responsive ovarian cancer patients.

Specific lysis of TAL at an A:T ratio of 50:l was 47.6 % on day 0 and declined to 3 % on day 15. This decrease was not related to the day-to-day variability of the assay, as shown by the NK activity of PBL

from the same patient and from normal donors tested in parallel on the same days.

The effect of i.p. C. parvum on the tumoricidal activity of TAM was investigated in six patients (Fig. 8b) . No evidence of augmentation of macrophage- mediated cytotoxicity was obtained throughout the 100-day observation period. In all four subjects in- whom macrophage-mediated cytotoxicity was measured between days 14 and 21 after the first C. parvum administration, a marked depression of the tumoricidal potential of TAM was observed at these times, specific lysis being 7.6 _+ 1.8 (mean f SD) compard to a pretreatment value of 25.5 _+ 5.5 (p <0.01). Subsequent evaluations 48 to 110 days after starting C. parvum therapy showed cytolytic activity levels similar to pretreatment values. The drop of cytotoxicity consistently observed between days 14 and 21 was not attributable to a failure of the assay to detect cytolysis on those particular occasions, as peripheral blood monocytes from the same patients or from normal donors tested in parallel on the same days showed no decrease in cytolysis values. Mac- rophage cytotoxicity at different A:T ratios for one of these subjects is presented in Figure 9. Specific lysis at an A:T ratio of 20:l was 32 % on day 0 and declined to 5 and 4 % on day 14 and 21. This decrease of macrophage cytotoxicity was not related to alterations in the sensitivity of the assay, because no such changes were detected with blood monocytes from the same subject or from normal donors tested in parallel on the same days (Fig. 9).

DISCUSSION

The results presented here confirm previous observations that i.p. C. parvum has limited anti-tumor activity in the palliative treatment of ovarian carcinomatous ascites resistant to chemotherapy (Webb et al., 1978). Five of eight subjects treated i.p. with this anaerobic coryneform showed a marked reduction of the ascites, and three of them experienced a complete disappearance of the effusion. The limited, but significant, anti-tumor activity of C. parvum injected i.p. in the ascites was confirmed by examination of the tumor cells present in the effusions. While peritoneal effusions were significantly af-

C o n t r o l monocytcs

444 MANTOVANI ET AL

fected by i.p. C. parvum, solid tumor masses were not modified by this agent under these conditions.

The main objective of the studies presented her- ein was to investigate alterations of the number and functional status of inflammatory cells present in ovarian carcinomatous ascites after i.p. treatment with C. parvum. Results obtained can be sum- marized as follows. The percentage of tumor cells decreased as early as 7 days after treatment, while polymorphs and macrophages increased. No consistent alterations in lymphoid cell percentages were observed. Although, to avoid subjective bias, coded smears were examined by two of us independently, identification of cell populations on a purely morphological basis may have led to erroneous conclu- sions, possibly with the exception of the easily iden- tifiable polymorphs. However, the increase in macrophages was also evident when recoveries of purified mononuclear phagocytes from ascitic tumors were considered (Fig. 5a). It is of interest that similar alterations in the tumor composition followed local injection of C. parvum in a murine carcinoma (Haskill et al., 1980).

I n vivo injection of C. parvum in mice enhanced NK activity, the effect being dependent on the route and time of administration and on the organ examined (Herberman et al., 1977; Oehler et al., 1978; Ojo et al., 1978; Flexman and Shellam, 1980). After C. parvum injection inhibition of NK cytotoxicity has also been described (Ojo et al., 1978; Flexman and Shellam, 1980). The dependence of the inhibition of NK lysis on the activation of suppressor cells by this agent has been the object of conflicting re- ports (Ojo ef al., 1978; Lotzova, 1980). In the present study, given the limited number of patients studied and the day-to-day variability of the assay, NK cytotoxicity in blood was not altered by i.p. C. parvum in a consistent pattern. NK cytotoxicity of untreated TAL was very low (RCI ~ 0 . 4 ) in three of four patients investigated and was not appreciably modified by C. parvum. In a fourth subject with an RCI of 0.8, a marked drop in cytotoxicity of TAL was observed as early as 7 days after the first treatment, whereas blood NK from the same patient was not inhibited (Fig. 7). These data suggest that inhibition of NK can occur in man after C. parvum treatment as observed in rodents (Ojo etal., 1978; Lotzo- va, 1980). TAL capable of suppressing NK have been found in human ascitic ovarian tumors (Manto- vani et al., 1980a) and it remains to be elucidated whether activation of suppressor cells occurs after i.p. C. parvum in humans.

Under certain experimental conditions at least, stimulation of NK activity by C. parvum in mice was short-lived, being detectable only a few days after treatment (Herberman et al., 1977; Oehler et al., 1978; Ojo et al., 1978; Flexman and Shellam, 1980). Therefore we cannot exclude that we missed an early, transient, stimulatory effect of C. parvum on TAL NK activity, because the earliest time tested in the present study was 7 days after treatment.

Intraperitoneal administration of C. parvum has been shown repeatedly to cause a long-lasting stimu-

lation of macrophage cytotoxicity in rodents (see for instance Ghaffar et al., 1975; Olivotto and Bomford, 1974). Human PBL exposed in vitro to C. parvum produce IF (Sugiyama and Epstein, 1978), an agent capable of enhancing human macrophage cytotoxicity (Jett et al., 1980; Mantovani et a[., 1980b, c ) . Morever, supernatants generated by co-culture of human PBL with anaerobic coryneforms augmented the cytolytic activity of human monocytes and inhibited their migration (Mantovani et al., 1980~; Tag- liabue et al., 1980). On the basis of these data, intraperitoneal injection of C. parvum in human ovarian ascites was expected to stimulate the tumoricidal activity of TAM. This prediction was not verified in the present study. Between 14 and 21 days following the first C. parvum treatment, a significant decrease in the tumoricidal activity of TAM was consistently observed, cytotoxicity returning to pretreatment values at later times. No such changes were observed with peripheral blood monocytes from the same patients. The mechanism(s) responsible for inhibition of ascites macrophage cytotoxicity by C. parvum remains to be elucidated, but it is noteworthy that, while this work in humans was in progress, Haskill and co-workers (1980) reported a similar inhibition of the direct and antibody-dependent cytotoxic activity of TAM from a murine carcinoma following intratumor administration of C. parvum. One could argue that transient depression of in vitro macrophage-mediated cytotoxicity is in fact the expression of an in vivo activation of this host defense mechanism and of its exhaustion within the tumor, but no experimental evidence supports this hy- pothesis. An alternative or complementary possibili- ty is that, 14-21 days after C. parvum treatment, macrophages are actively engaged in phagocytosing degenerating tumor cells and/or the injected anaeroboic coryneforms, and, at least under certain cirumstances, phagocytosis can inhibit the expression of macrophage cytotoxicity (Weinberg and Hibbs, 1977).

The mechanisms responsible for the limited but significant anti-tumor activity of i.p. C. parvum in ovarian ascites were not clarified by the results obtained in the present study. No correlation was observed between the pretreatment cellular composition of the ascites, or the functional status of NK cells and macrophages, and subsequent response to C. parvum therapy. Moreover, activation of NK andlor macrophage cytotoxicity was not found. Therefore, other, not mutually exclusive mechanisms, not specifically analysed in the present study, should be considered. Fibrosis can occur late after i.p. C. parvum in humans (Webb et al., 1978) and this was clinically suspected in some of the patients studied. However, it is extremely unlikely that fibrosis per se can account for the rapid decrease in tumor cell numbers and disappearance of ascites in the first weeks after treatment. The functional status of polymorphs which enter the ascites early after C. parvum was not investigated, but since these cells are capable of non-specific cytostasis on tumor cells (Korec et al., 1980), they may play a role in the limited anti-tumor activity of this agent in this human neoplasm.


Macrophage numbers were increased in peritoneal effusions after C. parvurn therapy, both in relation to other cell types and in terms of the absolute numbers recovered. Although macrophage cytotoxicity was not augmented by C. parvurn and was significantly depressed between days 14 and 21, the mod- ifications of the macrophage: tumor cell ratio induced in ascites could have in vivo relevance. In fact, the expression of human macrophage cytotoxicity in vitro is critically affected by the A:T ratio employed, stimulation of tumor cell proliferation being observed with low numbers of mononuclear phagocytes (Mantovani et al., 1979a, b) . Similar results have been reported with rodent effector cells. Thus, an increase in macrophage numbers could, at least partially, compensate for the drop in cytotoxicity observed between days 14 and 21 and permit the expression in vivo of some anti-tumor activity by this host defense mechanism.

In relation to the possible mechanisms of the anti- tumor activity of c. parvurn, it is noteworthy that no activation of direct and antibody-dependent intratumor macrophage cytotoxicity occurred after treatment with C. parvurn, of mice bearing a mammary carcinoma (Haskill et al., 1980). Experiments with diffusion chambers suggested that a humoral factor could mediate C. parvum-induced tumor inhibition (Haskill et al., 1980).

In the last few years there has been a considerable effort to develop immunotherapeutic approaches to human neoplasia, using non-specific immuno- modulatory compounds, such as C. parvurn, on an

empirical basis. In general, therapeutic results so far have been disappointing and, at best, marginal, as in the present study (Alexander, 1977; Terry and Windhorst, 1978). In this connection, the results reported here are of some interest, inasmuch as a marked stimulation of the cytotoxicity of TAM and, possibly, NK cells was expected on the basis of animal data, but was not detect in human ovarian ascites treated i.p. with C. parvurn. These findings em- phasize the need to evaluate agents as candidates for immunotherapy in man, for their effects on host defense mechanisms in human neoplasia. Carcinoma- tous ascites is a frequent event in advanced human ovarian neoplasms and provides an easily accessible source of tumor cells, macrophages and lymphoid cells in suspension. The present study indicates that ovarian ascites can be invaluable in assessing the effects of therapeutic agents on host defense mechanisms at an anatomical site directly involved by neoplasia.

ACKNOWLEDGEMENTS

This work was supported by “Progetto Finaliz- zato Controllo della Crescita Neoplastica” (No. 79.00643.96) from CNR, Rome, Italy, and laboratory studies were in part supported by grant l R O l CA 26824 from the National Cancer Institute, USA. We wish to thank Drs. C. Belloni, C. Bortolozzi, G. Bolis, M. D’Incalci, C. La Vecchia and P. Molteni for invaluable help in the collection of biological samples. We thank Dr. M. Recchia for guidance in the statistical analysis of the data.

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