Original Paper International Journal of Cell Cloning 9:511-520 (1991)

In Vivo Effects of Recombinant Human Interleukin 6, Alone or in Combination with Local Irradiation, on Tumor Growth in Lewis Lung CarcinomaBearing Mice Li Lu".4 Rong-Nian Shen".

8'

4 Hal E. Broxmeyer".

Departments of 'Medicine (Hematology/Oncology), bMicrobiology and Immunology, 'Radiation Oncology, and %he Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA

Keywords. rhIL-6

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Irradiation

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Lewislungcarcinoma

Metastases

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Mice

CFU-gm

Abstract. Lewis lung carcinoma (LLC)-bearing mice were used as a mouse model to evaluate effects of recombinant human (rh) interleukin frL) 6 and local X-irradiation

(LR) on the growth of primary tumors and lung metastases. Mice were inoculated S.C. with LLC tumor cells and then treated with rhIL-6 (100 ng/dose) S.C. twice a day (b.i.d.) for 5 days, beginning 6 days after tumor inoculation. LR (800 cGy) was administered to the site of the primary tumor 6 days after tumor inoculation and again 1 wk later. Mice were then observed for survival or sacrificed at day 21 after tumor inoculation to determine size of primary tumor, numbers and size of lung metastases, and other hematological parameters including numbers of granulocyte-macrophage progenitor cells (CFU-gm). The size of the primary tumor and numbers of lung metastases were reduced by rhIL-6. LR enhanced the antitumor effect of rhIL-6 significantly, while LR alone had only a slight antitumor effect. lkmor-associatedincreasesin peripheral blood, femoral marrow, splenic-nucleated cellularity, and marrow and splenic CFU-gm were reduced in mice treated with rhIL-6 plus LR. Prolonged survival time was observed only in tumor-bearing mice treated with rhIL-6 in combination with LR. The antitumor effects in vivo of rhIL-6 appear to be mediated indirectly as rhIL- 6 had no effect on proliferation of LLC cells in vitro as assessed by colony and 3H-thymidineincorporation assays. These studies suggest that rhIL- 6 may have therapeutic value in the treatment of certain malignancies, especially if used in combination with LR.

Introduction Cytokines have been used as immunomodulatory and therapeutic agents in Correspondence: Li Lu, M.D., Department of Medicine (Hematology/Oncology) and the Walther Oncology Center, Indiana University School of Medicine, 975 W. Walnut Street, IB 558, Indianapolis, IN 46202-5121, USA. Received February 12, 1991; provisionally accepted March 4, 1991; accepted for publication March 21, 1991. 0737-1454/91/$2.00/0 oAlphaMed Press

Effects of r h n - 6 in Tumor-Bearing Mice

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attempts to slow the growth of, or to eradicate malignant tumors in animals [l], in attempts to evaluate the potential usefulness of these molecules for human disease. For example, interleukin (IL) 2, macrophage colony-stimulating factor (M-CSF), tumor necrosis factor, interferon-gamma,and IL-6 have been used with varying degrees of efficacy against different solid tumors in animal models [2-81. IL-6 is a multi-functional cytokine with actions on many different cell types [9,lo]. The genes for both IL-6 and its receptor have been cloned [ll]. In the present report we have assessed the efficacy of recombinant human (rh) IL-6, alone and in combination with local irradiation (LR) to the site of the tumor, for effectiveness against the growth of Lewis lung carcinoma cells (LLC) in mice. The results demonstrate the antitumor activity of rhIL-6 in this animal model, an effect enhanced by combination treatment of mice with LR.

Materials and Methods Mice

Six-to eght-week-old female C57BU6.l mice were purchased from Jackson Laboratory (Bar Harbor, ME) and used in all experiments. Tumor cells

LLC cells were maintained by S.C. inckulation in C57BLl6.l female mice. They produce metastases, preferentially in the lungs, as described previously [3,5]. Briefly, primary tumors were isolated from mice and minced with phosphate-buffered saline (PBS). Single cells were washed three times and resuspended in RPMI 1640 (Whittaker M.A. Bioproducts Inc., Walkersville, MD).The viability of LLC cells was >90% as assessed by the trypan blue dye exclusion method. Viable LLC cells (5 x 10’ in 0.1 ml) 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 rhn-6 (specific activity 5 X lo6Ulmg) which was measured in cultures of human lymphoblastoidCESS cells, and was a generous gift from Drs.Steven Gillis and David Urdal, Immunex Corporation, Seattle, WA. rhn-6 was administered s . ~ twice . a day (bid.) to both limbs (around the tumor when injection was given in the limbs with tumor) for 5 days beginning on day 6 after tumor inoculation. Local X-irradiation (LR) was performed on day 6 and 13 after tumor inoculation. Mice were immobilized (without anesthesia) on a specially designed board. The primary tumors were locally irradiated (after shielding the mice except for the area of the primary tumor) with 800 cGy of X-ray at a dose of 239.5 cGylmin using a Siemens Stabilipan X-ray machine (Siemans Medical Systems, Inc., Iselin, NJ; 250 KV, 1.0mm Cu filter, 2.1, Cu HVL, SSD 23 cm) as described previously [3]. The mice were then observed for survival time, or sacrificed 21 days after tumor inoculation to determine the size of primary tumors and the number and size of lung metastases. Hematologicalparameters assessed included femoral marrow and splenic granulocyte-macrophage progenitor cells (CFU-gm), and nucleated cellularity of bone marrow (BM), spleen and peripheral blood (PB). Measurement of Primary Tmor and Lung Metastases The size of primary tumor was measured by 2 perpendicular diameters calculated

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A) Lung Metastases

B ) Primary Tumor

Numbers

50

t

40

4

30

3

20

2

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1

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2mnq 1 W . q 1 0 n p W". am nq 1wnp 10 nq I I I *lL6

rnlL.8

Treatment

Fig. 1. Influence of rhIL-6 on A) number of lung metastases and B) size of primary tumor in LLC-bearing mice. Mice were inoculated S.C. with LLC tumor cells on day 0. Beginning on day 6, mice received to both limbs (around the tumor when injection was given on the limb with tumor) treatment with saline, rhIL-6 at 10 - 200 ng/dose, S.C. b.i.d. for 5 days. Mice were sacrificed 21 days after tumor inoculation. Results are expressed as mean f SEM for 3 mice/group. Mann-Whiney test and Student's t test were used to evaluate results for both lung metastases and primary tumor. Significant difference from mice given saline: a = p < O.ooO1; b = p < 0.01.

Measurement of Primary Tumor and Lung Metastases The sue of primary tumor was measured by 2 perpendicular diameters calculated by the formula, v = 0.4 (abz) cm3,where b was the smaller tumor, as reported previously [3]. The lungs were stained by infiltration with 15% India ink solution and fixed in Feketes solution and assessed using a magnifying lens (8X) [3]. Colony Assay BM (1 x lOVml), spleen (1 X 106/ml)and LLC (1 X 103/ml)cells were cultured in 0.3% semisolid agar culture medium (Difco, Detroit, MI) with 10%fetal calf serum (FCS; Hyclone, Logan, UT)and 10%medium conditioned ty pokeweed-mitogen-stimulatedmouse spleen cells (PWM-SCM) [5]. Colonies ( >50 cells) and clusters (5 to 50 cells) of granulocytes and macrophagesor LLC cells were scored after 5 to 7 days of incubation at 5% C02, 20% 0,. 3H-thymidine (3H-Tdr) Incopomtion Assay DNA synthesis was measured by 3H-Tdruptake as described [El. Briefly, LLC cells were suspended at 105 celldwell in 96-well plates and incubated with rhIL-6 at 1 pglml, 100ng/ml, 10 ng/ml or 1 ng/ml at 5%C02,20% O2and 37°C for 24 and ?2 h. One pCi/well of 3H-Tdr added 4 h before harvesting and uptake of 3H-Tdr into cells was measured by liquid scintillation counting.

Effects of rhIL - 6 in Tumor-Bearing Mice

A) Lung Metastases Numbers

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El) Primary Tumor ern’ lo

I

Treatment

Fig. 2. Influence of rhIL-6, alone and in combination with local irradiation, on A) numbers of lung metastases and B) size of primary tumor in LLC-bearing mice. Mice were inoculated with LLC tumor cells as described in the legend to Figure 1. Beginning on day 6, mice received rhIL-6 at 100 ng/dose, S.C. b i d . for 5 days, local irradiation (LR, 800 cGy at the 6th day and then again 1 wk later to the site of the tumor) or rNL-6 plus LR. The average results of mice sacrificed 21 days after tumor inoculation from 3 experiments are expressed as mean f SEM. Numbers in parentheses represent the total number of mice per group. Significant change from saline control designated as: a = p < O.OOO1; b = p < 0.01; c = p c 0.05. Statistical Analysis Mice in each group or cell samples were individually assessed in each experiment. The probability of significant differences between groups or samples was determined by the Student’s t test and Mann-Whitney test. Statistics for survival times were calculated using the Log rank test [13].

Results Influence of rhIL-6, With or Without L&, to the Site of the Primary Ittmor, on Lung Metastases and Primary Tumor Mice were administered rhIL-6 at 10, 100 and 200 ng S.C. b i d . for 5 days starting 6 days after tumor inoculation. As noted in Figure 1, rhIL-6 at 100 ng, but not at 10 ng, significantly decreased numbers of lung metastases and size of the primary tumor. Administration of rhIL-6 at dosages of 200 ng were no more effective than at dosages of 100 ng. In an effort to assess the possibility that LR at the site of the tumor might enhance the systemiceffects of rhIL-6, LLC-bearing mice were treated with saline as a control, LR, rhIL-6 (100 ng per dose), or the

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Table I. Effect of rhIL-6, alone or in combination with LR, on numbers and size distribution of lung metastases in LLC-bearing mice Size of lung metastases (mm') Treatment

No. lung metastases

In vivo effects of recombinant human interleukin 6, alone or in combination with local irradiation, on tumor growth in Lewis lung carcinoma-bearing mice.

Lewis lung carcinoma (LLC)-bearing mice were used as a mouse model to evaluate effects of recombinant human (rh) interleukin (IL) 6 and local X-irradi...
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