Comparison of responsiveness to cancer development and anti-cancer drug in three different C57BL/6N stocks

Abstract

In our efforts to understand the systemic features of tumors, the importance of animal models is increasing due to the recent growth in the development of immunotherapy and targeted therapies. This has resulted in increased attention towards tumor animal models using C57BL/6N, which are mainly used in immunological studies. In this study, the C57BL/6NKorl stock and two other commercial stocks (C57BL/6NA and C57BL/N6B) are evaluated by comparing the occurrence of tumors using the syngeneic model; furthermore, we compare the response to anti-cancer drugs in the syngeneic model by evaluating survival, growth of tumors, proliferation and molecular biology analysis. In the syngeneic model using LLC (Lewis lung carcinoma) cells, the survival of mice and growth of the tumor showed a better response in the C57BL/6NKorl stock, and was dependent on the cell concentration of the dosing tumor, as compared to the other C57BL/6N stocks. However, the Ki-67 staining showed only little difference in cell proliferation within the tumor tissue each mouse stocks. Comparing the sensitivity to anti-cancer drug by examining changes in growth, volume and weight revealed that cisplatin treatment for tumor-bearing C57BL/6NKorl was more dependent on concentration. The Ki-67 staining, however, showed no difference among the C57BL/6N stocks after cisplatin treatment. The expressions of p27 and p53 tumor suppressor proteins, caspase-3 and Bax showed dose-dependent increase after exposure to cisplatin, whereas the expression of Bcl-2 was reduced in a dose-dependent manner. Furthermore, the expressions of MMP-2 and VEGF involved in metastasis, as well as inflammatory genes IL-1β, IL-6 and IL-10, showed dose-dependent decrease in tumor tissue after cisplatin exposure. Differences observed among the C57BL/6N stocks were not significant. Taken together, our studies reveal that C57BL/6NKorl has the potential of being a useful biological resource established in Korea, as it does not differ from the two commercially available C57BL/6N stocks when considering response to tumor generation and sensitivity to anti-cancer drugs using the syngeneic tumor model.

Keywords: C57BL/6N; C57BL/6NKorl; Cisplatin; Syngeneic tumor model.

Fig. 1

Comparison of the three C57BL/6N stocks on survival rate of mouse in LLC tumor cell transplant model. Four different numbers (5x10⁴, 1 x 10⁵, 5 x 10⁵ or 1 x 10⁶ cells) of LLC cells were subcutaneous injected in C57BL/6NKorl (a), C57BL/6NA (b), and C57BL/6NB (c), respectively. Each group consisted of 8 mice. The mouse survival was evaluated as described in Materials and Methods. Data represents the mean ± S.E.M of n = 8/group

Fig. 2

Comparison of the three C57BL/6 N stocks on tumor growth of mouse in LLC tumor cell transplant model. Four different numbers (5x10⁴, 1 x 10⁵, 5 x 10⁵ or 1 x 10⁶ cells) of LLC cells were subcutaneous injected in C57BL/6NKorl (a), C57BL/6NA (b), and C57BL/6NB (c), respectively. The tumor mass was evaluated at every 2 days. d Optic observation of growth tumors from each stock mice 27 days after LLC (indicated cells number/200 μl/body) transplantation. Each group consisted of 8 mice. The mouse growth was evaluated as described in Materials and Methods. Data represents the mean ± S.E.M of n = 8/group (*P < 0.05 versus No group)

Fig. 3

Comparison of cisplatin efficacy in inoculated LLC tumor cells among the C57BL/6NKorl, C57BL/6NA and C57BL/6NB. Three different concentration (100 μg/kg (LCP), 1 mg/kg (MCP) or 5 mg/kg (HCP)) of cisplatin were intra peritoneal injected in LLC cell bearing mice, C57BL/6NKorl (a), C57BL/6NA (b), and C57BL/6NB (c). The tumor mass was evaluated at every 2 days. Middle paragraph presents optic observation of growth tumors volume from each stock mice at 24 days after LLC transplantation. Bottom paragraph represents optic observation of isolated tumors mass. Isolation of tumor mass from each stock mice at 24 days after LLC transplantation. Each group consisted of 8 mice. Data represents the mean ± S.E.M of n = 8/group (*P < 0.05 versus LLC+Ve group)

Fig. 4

Comparison of p27 and p53 protein expression by cisplatin in tumor samples from the LLC cell bearing C57BL/6N mice stocks. Relative protein expression levels were measured using the western blotting. a, b, and c plots present the relative protein expression level of tumor tissue from cisplatin-treated tumor bearing C57BL/6NKorl (a), C57BL/6NA (b), and C57BL/6NB (c) stocks, respectively. Data represents the mean ± S.E.M of n = 8/group (*P < 0.05 versus LLC+Ve group)

Fig. 5

Determination of cisplatin-induced apoptosis in tumor tissue collected from LLC cell bearing C57BL/6N mice stocks using comparing the Bax/Bcl-2 protein ratio. Total lysates were prepared from the tumor tissues of cisplatin-treated tumor bearing C57BL/6NKorl (a), C57BL/6NA (b), and C57BL/6NB (c) mice of each group, as described in Materials and Methods. A total of 50 μg of protein per sample was immunoblotted with antibodies for each protein. Three samples were assayed in triplicate by western blotting. Data represents the means ± S.E.M of three replicates. *P < 0.05 versus LLC+Ve group