Int. J . Cancer: 47, 143-147 (1991) 0 1991 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Pubiication de I'Union Internationale Contre le Cancer

ANTI-TUMOR EFFECTS OF RECOMBINANT HUMAN MACROPHAGE COLONY-STIMULATING FACTOR, ALONE OR IN COMBINATION WITH LOCAL IRRADIATION, IN MICE INOCULATED WITH LEWIS LUNG CARCINOMA CELLS Li LLJ'.~,~, Rong-Nian SHEN'*374,Zhong-Hua LIN',~,Sharon L. AUKERMAN~, Peter RALPH~ and Hal E. BROXMEYER'.~~~ Deparmnts of 'Medicine (HematologylOncology),2Microbiology and Immunology and 3Radiation Oncology; 4The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202; and Departments of 5 P h a m ~ ~ land ~ g 6Cell y Biology, Cetus Corporation, Emeryville, CA 94608, USA. Recombinant human (rhu) macrophage colony-stimulating factor (M-CSF) was evaluated for efficacy, either alone or in combination with local X-irradiation (LR), in mice inoculated subcutaneously (s.c.) with Lewis lung carcinoma (LLC) cells. The size of the primary tumor and numbers of lung metastases, 21 days after tumor inoculationand I 5 days after the start of treatment, were reduced by 87% in tumor-bearing mice treated with 20 pg/dose M-CSF S.C. twice a day for 5 days. LR (800 cGy) to the tumor once a week for 2 weeks had a moderate anti-tumor effect and enhanced the anti-tumor effect of M-CSF. Hematological parameters, including nucleated cellularity in peripheral blood, femoral marrow, spleen and peritoneal exudate, as well as marrow and splenic granulocyte-macrophage progenitor cells, and numbers of splenic Thyl.2+ cell and peritoneal mast cells, were perturbed in LLC-bearing mice, and were influenced by treatment with M-CSF and L R Treatment with M-CSF plus LR, but not with either agent alone, was associated with a significant, although slight, enhancement in survival time for LLC-bearing mice. Inability to obtain a better survival-enhancingeffect appeared to be related to the limited treatment, since the anti-tumor effects of M-CSF were more notable early on in disease progression and were related to the dose of M-CSF used. The effects of M-CSF were most probably indirect ones on the host immune system. M-CSF, in combinationwith LR. may be of benefit in the treatment of human tumors that have metastatic potential.

Treatment of malignant tumors, especially tumors which metastasize, continues to present a clinical challenge. Intensive chemotherapy and radiotherapy include myelosuppression or immunosuppression, or both, which require substantial supportive care, and patients die as a result of either advanced cancer or bone-marrow failure and secondary infection. Thus, it is important to develop efficient but less toxic methods for treating patients with malignant disease. Cytokines are currently being evaluated as immunomodulatory and therapeutic agents in the clinic (Gillis et al., 1986; Broxmeyer and Vadhan-Raj, 1989). To this end they have been assessed for possible anti-tumor effects in animal tumor models (Shen et al., 1990). One cytokine that may be of use is the macrophage colony-stimulating factor (M-CSF or CSF-I), a glycoprotein required for the growth and differentiation of macrophage progenitors as well as the survival of mature macrophages (Das et al., 1982; Broxmeyer and Williams, 1988; Ralph et al., 1989; Broxmeyer et al., 1990). The human cDNA for the M-CSF gene has been cloned and sequenced (Kawasaki et al., 1985; Wong et al., 1987). Native and recombinant human and mouse M-CSF are biologically active when administered to mice (Broxmeyer et al., 1987a,b,c, 1988; Williams et al., 1987; Hume et al., 1988; Chikkappa et al., 1989), rats (Ulrich et al., 1990) and non-human primates (Munn et al., 1990). A natural form of M-CSF, human urinary CSF, has been used in the clinic (Komiyama et al., 1988) and M-CSF has been shown to reduce the incidence of spontaneous metastases in mice inoculated with B16 melanoma cells (Hume et al., 1989). In the present report, the effects of recombinant human (rhu) M-CSF were assessed, alone and in combination

with local X-irradiation (LR), in mice bearing Lewis lung carcinoma (LLC) tumor. The results demonstrate that rhuM-CSF has anti-tumor effects both on the primary LLC tumor and on the metastatic potential of these cells and that this effect, which is dose-dependent and time-related, is enhanced when local irradiation is applied to the site of the primary tumor. MATERIAL AND METHODS

Mice Female C57BL/6J mice, 6-8 weeks old, purchased from Jackson Laboratory, Bar Harbor, ME, were used in all experiments. Tumor cells LLC cells were maintained by S.C. inoculation in C57BLi6J female mice. They produce metastases, preferentially in the lungs. Primary tumors were isolated from mice and minced with phosphate-buffered saline (PBS). Single cells were washed 3 times and resuspended in RPMI 1640. Viability of LLC cells was >90% as assessed by the Trypan blue dye exclusion method. Viable LLC cells (5 X lo5 in 0.1 ml medium) were inoculated S.C. into the right hind limb. Treatment Six days after tumor-cell inoculation, mice were randomly divided into several groups for treatment. Mice were treated with rhu M-CSF [long clone ending at 223 amino acids produced in E . coli (Halenbeck et al., 1989); specific activity 4.7 X lo7unitsimg; lot DP403, Cetus, Emeryville, CAI given as kg/dose s.c., twice a day (b.i.d.), to both limbs in turn (around the tumor when injection was given in the limbs with tumor) for 5 days. Mice were immobilized (without anesthesia) and tumors were locally irradiated (after shielding the mice except for the area of the primary tumor) with 800 cGy of X-rays at a dose rate of 239.5 cGyImin using a Siemens Stabilipan X-ray machine (250 KV, 1.O mm Cu filter, 2.1, Cu HVL, SSD 23 cm) 6 and 13 days after tumor inoculation (Shen et al., 19886). Mice were observed for survival time, or killed to determine the size of primary tumor and the number and size of lung metastases. Other hematological parameters evaluated included growth of femoral marrow and splenic hematopoietic progenitor cells, and cellularity of peripheral blood, blood

7T0whom correspondence and reprint requests should be sent, at the Walther Oncology Center, Indiana University School of Medicine, Medical Research and Library Building, 975 W. Walnut Street, Room 558, Indianapolis, IN 46202-5121, USA. Abbreviations: rhu, recombinant human; M-CSF, macrophage colony stimulation factor; LR, local irradiation; LLC, Lewis lung carcinoma.

Received June 22, 1990 and in revised form September 17, 1990.

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LU ET AL.

monocytes, bone marrow, spleen, peritoneal exudate, peritoneal exudate macrophages and splenic Thy 1.2 cells. +

Measurement of primary tumor and lung metastases The size of primary tumors was measured by 2 perpendicular diameters calculated by the formula, v = 0.4 (ab2) cm3, where b was the smaller diameter. Lungs were stained by infiltration with 15% solution of Indian ink, fixed in Fekete’s solution and checked at X 8 magnification (Shen et al., 1988b). Colony assay Bone-marrow cells ( 105/ml)and spleen cells ( 106/ml) were cultured in 0.3% semi-solid agar culture medium (Difco, Detroit, MI) with 10% fetal calf serum (Hyclone, Logan, UT) and 10% medium conditioned by pokeweed-mitogen-stimulated mouse spleen cells (PWMSCM) (Lu et al., 1985). Colonies (>50 cells) and clusters 5 to 50 cells) of granulocytes and macrophages were scored after 5 days of incubation at 5% c o * , 5% 0 2 .

TABLE I - EFFECT OF RECOMBINANT HUMAN M-CSF. ALONE OR IN COMBINATION WITH LR. ON THE GROWTH OP PRIMARY TUMORS AND THE NUMBER OF LUNG METASTASES IN LLC-BEARING MICE Treatment

Saline LR M-CSF M-CSF

+ LR

N

Number of lung metastases

18 13 18 14

28.8 2.4 14.3 3.4 (-50)2 9.3 0.8 (-68)’ 3.9 t 0.8 (-87)’

* * *

Primary

tumor (cm3)

* *

6.5 0.06 2.8 2 0.6 (-57)’ 2.8 0.4 (-57)‘ 0.8 f 0.2 ( - 8 7 ) ’

Mice were inoculated S.C. with LLC tumor cells on day 0. Beginning on day 6, mice received at both limbs in turn (around the tumor when injection was given on the limbs with NmoI) treatment with saline, local .wadiation (LR, 800 cGy once a week for 2 weeks to the site of the tumor), recombinant human M-CSF (20 Fgldose, S.C. bid for 5 days), or M-CSF plus LR. Mice were sacrificed 21 days after tumor inoculation. The average results are expressed as mean 2 SEM from 3 experiments. Numbers in parentheses represent the percentage change from saline control. “N” represents the number of mice. Mann-Whitney test and student’s t test were used respectivelyto evaluate results for lung metastases and primary tumor. -‘p < O.ooO1; -2p < 0.01.

Analysis of T-lymphocytes Red blood cells in the spleen were lysed with distilled water and spleen cells analyzed by a Coulter Epics 753 Dye Laser Flow Cytometry System with monoclonal Anti-Thy 1.2 (Becton-Dickinson, Mountain View, CA) as described previously . were incubated for 20 min at 4°C (Shen et al., 1 9 8 8 ~ )Cells with 4 p i anti-thy 1.2/FITC per lo6 cells, washed 3 X and kept at 4°C until analyzed. Peritoneal exudate cells Mice were injected i.p. with sterile pyrogen-free saline and the saline containing cells was withdrawn from the peritoneum. Statistics analysis Three or more mice in each group were individually assessed in each experiment. The probability of significant differences between groups or samples was determined by Student’s t-test and Mann-Whitney test. Statistics for the survival time were calculated using the Log rank test (Anderson et al., 1980). RESULTS

Effects of recombinant human M-CSF andlor local irradiation (LR)on primary tumor and lung metastases in LLC-bearing mice In preliminary experiments, 20 pgldose b.i.d. of M-CSF were given i.v. or S.C. to the non-tumor-bearing limb or to both limbs of LLC-bearing mice starting at day 6 after tumor innoculation. Numbers of lung metastases were reduced in mice treated with M-CSF, and results were better in mice treated with M-CSF, S.C. to both limbs (63% reduction) compared to i.v. (5% reduction) and S.C. to the non-tumor-bearing limb (20% reduction) (data not shown). This was consistent with a recent report that injection of other lymphokines close to the tumor is an effective maneuver (Vaage, 1988; Forni et al., 1989). Thus, for the rest of the studies, treatment was started at day 6 S.C. to both limbs in turn, with injections close to the primary site when injection was given to the tumor-bearing limb. Table I (average of 3 experiments) gives results assessing the effects of M-CSF, alone or in combination with LR. Numbers of lung metastases and size of the primary tumor were reduced by 87% in tumor-bearing mice treated with 20 pgldose b.i.d. M-CSF in combination with LR, while treatment with either LR or M-CSF was less effective. Figure 1 shows representative pictures of actual lung metastases in mice treated with saline, or M-CSF, or LR, alone and in combination. The size and number of the lung metastases were reduced by treatment. Hematological parameters Inoculation of mice with LLC (saline group) was associated 21 days later with increased levels of nucleated peripheral

FIGURE 1 - Representative lung metastases in LLC-bearing mice 21 days after tumor inoculation. Six days after tumor-cell inoculation mice were treated with saline (a). local irradiation (LR) on the tumor with 800 cGy weekly for 2 weeks (b), M-CSF at 20 p,g/ dose S.C. b.i.d. for 5 days ( c ) , M-CSF plus LR (d).

blood, bone-marrow, splenic and peritoneal exudate cells, as well as absolute numbers of femoral and splenic granulocytemacrophage (CFU-GM) progenitor cells (Table 11). After treatment with M-CSF plus LR, some of the parameters (nucleated femoral marrow and peritoneal exudate cellularity, as well as marrow and splenic CFU-GM numbers) were close, or equal, to the values of the non-tumor-bearing normal mice. Peritoneal exudate macrophage differentials ranged from 86 -t 5 to 91 2 2% in all categories, so were consistent with changes noted in all groups for total peritoneal exudate cells. Numbers of peripheral blood monocytes and splenic Thy 1.2+ cells were increased above those of the non-tumor-bearing controls by treatment with M-CSF and LR, alone and in combination. Influence of M-CSF, alone or in combination with LR,on survival of LLC-bearing mice As shown in Figure 2, the survival time of mice was slightly,

145

ANTI-TUMOR EFFECTS OF HUMAN M-CSF IN LLC MICE

Table U - EFFECT OF rhuM-CSF. ALONE OR IN COMBINATION WITH LOCAL IRRADIATION (LRL ON HEMATOLOGICALPARAMETERS IN LLC-BEARINGMICE

Peripheral blood

Normal (no tumor) Saline LR M-CSF M-CSF

+ LR

6.2 f 1 15.9 f 3 ( + 156)' 13.5 f 1 ( + 118)' 16.3 f 3 (+ 163)' 12.7 f 1 ( + 105)'

0.37 0.24

Bone marrow

f 0.06

f 0.05

( - 35)

0.31

-t

0.02

( - 16)

1.14 f 0.02 ( + 208)' 1.87 f 0.15 ( 405Y

+

48 f 2 134 f 15 ( + 179)' 84 -t 12 (+ 75)* 73 f 26 (+ 52)3 54 f 3 [+ 13)

12.5 & 1 18.5 ? 4 ( + 48)3 18.3 f 2 ( + 46)3 11.3 & 4 ( - 10) 11.8 k 3

Peritoneal exudate

Spleen

32 f 1 92 f 39 ( + 188)' 6025 ( + 188)' 91 19 (+ 184)' 66 & 9 (106)'

*

6 f 2 84 f 25 ( + 1300)' 18 -t 3 ( + 200)' 29 f 2 (+ 383)' 15 f 3 ( + 150)'

3.3 f 0.1 7.9 f 1.1 ( f 139)* 7.1 2 0.1 (f115)' 4.9 f 0.1 (f48) 3.9 f 0.2 (i-18)

13.6 f 0.8 12.8 f 3.2 (-6) 20.0 f 11 ( + 47)3 38.7 f 17 ( f 163)' 20.7 f 1 + 52)3

Mice were inoculated S.C. with LLC cells on day 0 and treated as described in the legend to Table I. Mice were killed on day 21. Results are expressed as mean f SEM of 3 mice for each group from 1 of 3 representative experiments. Numbers in parentheses designate percent change from normal non-tumor-bearingmice.-'Signifjcant changes compared to non-tumor-bearing control. p < O.OOOl;-zp < 0.01;-3p < 0.05.

-sh 0

2-

60-

m

5 40-

- saline

8

-- -- saline+LR

20 -

___

M-CSF

i.,

M-CSF *LR

3 -

i

0 10

14

18

22

26

30

34

38

42

46

50

Days after inoculation with Lewis Lung Carcinoma (LLC) cells S.C. with and without treatment of M-CSFalone or in combination with local radiation (LR)

FIGURE 2 - Survival time of LLC-bearing mice. Six days after tumor-cell inoculation, mice were treated with saline or M-CSF at 20 p,g/dose S.C. b.i.d. for 5 days, or with M-CSF and local irradiation. Ten mice were evaluated for each group.

but significantly (p < O.OOOl), prolonged by treatment with 20 pg/dose b.i.d. of M-CSF for 5 days in combination with LR (median survival time = 39 days), but not by treatment with either M-CSF (median survival time = 33.5 days) or LR (median survival time = 27.5 days) (1 representative of 2 similar experiments). In order to evaluate the reasons for the death of treated mice which had shown a drastic decrease in tumor size and in numbers of lung metastases by week 3 (Table I, Fig. l), mice were treated with LR, 0.1 to 20 pg/dose b.i.d. of M-CSF or LR plus 20 Fgldose b.i.d. of M-CSF and were assessed twice a week, until death, for size of primary tumor (Fig. 3) and at death, for number of lung metastases (Table In). LR and M-CSF were efficacious in decreasing the size of primary tumor. This action was most effective at weeks 2 4 and was not as obvious by the 5th week. This is most apparent from the titration studies with M-CSF (Table 111). By the 5th week, only mice treated with 20 Fgldose b i d . of M-CSF showed a significant decrease in size of primary tumor (Fig. 3) and number of lung metastases (Table 111), whereas concentrations of M-CSF as low as 1 pg/dose b i d . had significant suppressive effects on primary tumor size as early as 2 weeks after tumor inoculation. The influence of increasing concentrations of M-CSF on numbers of lung metastases is depicted in Table 111. DISCUSSION

M-CSF treatment resulted in anti-tumor activity with con-

2 -

1 -

n 1

2

3

4

5

Weeks

FIGURE3 - Effect of recombinant human M-CSF, alone or in combination with local irradiation, on the growth of primary tumors in LLC-bearing mice during their life-time. Mice were inoculated S.C. with LLC tumor cells on day 0 and treated as described in the legend to Table 1. The size of primary tumor was measured twice a week until the mice died. Results are for one of 2 similar experiments. There were 1&13 mice per group. If an animal died before week 5 , the value obtained at death was calculated into the results of the week of death, but was also calculated into the average for each succeeding week. Thus, the N value is the same for each week. "Significance values compared to mice given saline, p < O.OOO1; "p < 0.01; 'p < 0.05.

comitant decreases in the size of the primary LLC tumor, as well as decreases in the number and size of lung metastases. These effects were M-CSF dose-dependent, and enhanced in most cases by LR to the site of the primary tumor. The combination of treatments resulted in significant, although slight, prolongation of the survival time of LLC-bearing mice. It is possible that enhancement of survival time will require prolonged administration of M-CSF, perhaps given more frequently and at higher doses. This possibility is suggested by the results in which the best effects were noted in mice given the highest dosages of M-CSF, and by the fact that the anti-tumor effects were most apparent at an early stage of tumor development while the M-CSF was being given. The anti-tumor effect of M-CSF, which has been noted by others for B16 melanoma (Hume ef al., 1989) most probably reflects an influence on the host immune system. Mice bearing

146

LU ET AL.

TABLE I1I - EFFECT OF DIFFERENT CONCENTRATIONS OF RECOMBINANT HUMAN M-CSF ON THE GROWTH OF PRIMARY TUMORS AND NUMBER OF LUNG METASTASES IN LLC-BEARING MICE

Treatment

Saline

M-CSF 20 Pg M-CSF 10 Fg M-CSF 1 Fg M-CSF 0.1 Pg

Primary tumor (cm')

5.2 2.1 5.5 5.1 5.6

1.3 0.8 (-60)' f 0.1 ( + 6 ) f 0.9 (-2) 2 1.0 (+8) ?

L

Number

lune metastases

41.3 L 2.4 9.1 f 0.9 (-78)' 26.6 L 5.1 (-35)' 34.7 f 1.8 (- 15) 34.2 f 2 (- 17)

Mice were inoculated S.C. with LLC tumor cells on day 0. Beginning on day 6, mice received treatment with saline or rhuM-CSF at indicated doses S.C. to each limb in turn b.i.d. for 5 days. The size of the primary tumor was measured twice a week until mice died, for up to 5 weeks. The number of lung metastases was scored after mice died. All mice were assessed by 5 weeks. Results are expressed as mean ? SEM for the average of 10mice per group from a total of 2 experiments. The results include values from all mice that died of disease by week 5 , or earlier, or were killed at week 5 . The Mann-Whitney test and the Student's 2-test were used res ec tively to evaluate data for lung metastases and primary tumor.-'p < O.OOO1;- 2p