A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation

Abstract

Background: Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation on tumor growth.

Methods: Three-month old CBA/Lac mice were used in the experiments. Mice were injected with CP and human dsDNA preparation. The percentage of mature dendritic cells (DCs) was estimated by staining of mononuclear cells isolated from spleen and bone marrow 3, 6, and 9 days later with monoclonal antibodies CD34, CD80, and CD86. In the next set of experiments, mice were given intramuscularly injections of 1-3 × 105 tumor cells. Four days later, they were injected intravenously with 6-6.7 mg/kg Dox and intraperitoneally with 100-200 mg/kg CP; 200 mkg human DNA was injected intraperitoneally after CP administration. Differences in tumor size between groups were analyzed for statistical significance by Student's t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups.

Results: The conducted experiments showed that combined treatment with CP and dsDNA preparation produce an increase in the total amount of mature DCs in vivo. Treatment of tumor bearers with preparation of fragmented dsDNA on the background of pretreatment with Dox plus CP demonstrated a strong suppression of tumor growth in two models. RLS, a weakly immunogenic, resistant to alkalyting cytostatics tumor, grew 3.4-fold slower when compared with the control (p < 0.001). In experiment with Krebs-2 tumor, only 2 of the 10 mice in the Dox+CP+DNA group had a palpable tumor on day 16. The cytotoxic index of leucocytes was 86.5% in the Dox+CP+DNA group, but it was 0% in the Dox+CP group.

Conclusions: Thus, the set of experiments we performed showed that exogenous dsDNA, when administered on the background of pretreatment with Dox plus CP, has an antitumor effect possibly due to DC activation.

Figure 1

Time course of Krebs-2 tumor growth in mice (mean ± SEM). Time course of Krebs-2 tumor growth in mice (mean ± SEM). Mice received 200 mg/kg CP and human DNA at a total dose 4.5-6 mg. After this treatment, one group of mice was pre-immunized with Krebs-2 tumor antigens by a s.c. injection of 20 × 10⁶ repeatedly thawed-frozen tumor cells. The control group was injected with saline. Every group consisted of 10 mice. 10⁶ Krebs-2 tumor cells were challenged i.m. after the treatment. Immunization enhanced the suppressive effect on tumor growth [31].

Figure 2

Time course of maturation of mouse DCs from spleen (A) and bone marrow (B) after treatment with CP and dsDNA preparation (mean ± SEM). Time course of maturation of mouse DCs from spleen (A) and bone marrow (B) after treatment with CP and dsDNA preparation (mean ± SEM). 0 represents the number of mature DCs in untreated mice. Mice were injected with CP 200 mg/kg and 200 mkg of human dsDNA preparation 1 day (on the day of CP injection), 3, 4, and 5 days after CP treatment. Three, 6, and 9 days later, the fraction of MNCs was isolated from spleen and bone marrow. Every group consisted of 4-6 mice. The experiment was repeated twice.

Figure 3

Time course of RLS tumor growth in mice that received combined treatment with Dox, CP, and dsDNA preparation (mean ± SE). Time course of RLS tumor growth in mice that received combined treatment with Dox, CP, and dsDNA preparation (mean ± SE). Mice were given i.m. injections of 10⁵ RLS-40 tumor cells. Four days later, they were injected i.v. with 6.7 mg/kg Dox and i.p. with 100 mg/kg CP; 200 mkg human DNA was injected i.p. after 30 min, then 2 and 3 days after CP administration. The control group was injected with PBS. Every group consisted of 10 mice.

Figure 4

Time course of Krebs-2 tumor growth in mice treated with Dox, CP, and dsDNA preparation (mean ± SE). Time course of Krebs-2 tumor growth in mice treated with Dox, CP, and dsDNA preparation (mean ± SE). Mice were given 3 × 10⁵Krebs-2 tumor cells injected i.m.. Four days later, they were administered i.v. 6 mg/kg Dox and i.p. 200 mg/kg CP; 200 mkg human DNA was administered i.p. 30 min after CP, also 2, 3, and 5 days after it. The control group was injected with PBS. Every group consisted of 10 mice. The experiment was done in triplicate.