Administration of embryonic stem cells generates effective antitumor immunity in mice with minor and heavy tumor load

Abstract

The history of immunizing animals with fetal tissues to generate an antitumor response dates back a century ago. Subsequent reports supported the idea that vaccination with embryonic materials could generate cancer-specific immunity and protect animals from transplantable and chemically induced tumors. In our study, we found C57 BL/6 mice vaccinated with embryonic stem cells (ESCs) received obvious antitumor immunity, which protected them from the formation and development of lung cancer. Furthermore, we investigated the antitumor effects of administration of ESCs in mice with minor and/or heavy tumor load. The tumor growth was monitored, the proliferation of lymphocytes and secretion of cytokines were examined, and finally the tissue sections were approached by immunohistochemical and apoptosis staining. The results suggested that mice injected with ESCs received obvious tumor inhibition and retardation due to significant lymphocyte proliferation and cytokine secretion, which help to rebuild the host's immunity against cancer to some extent and comprise the main part of antitumor immunity. Moreover, mice with minor tumor load received stronger antitumor effect compared with mice with heavy tumor load, may be due to relatively intact immune system. Thus, besides their function as prophylactic vaccines, administration of ESCs could be a potential treatment for cancer, which obviously prevent and control the proliferation and development of malignant tumors.

Fig. 1

Antitumor immunity induced by administration of ESCs. a Scheme of ESCs immunization and tumor inoculation. Mice in the vaccine group received subcutaneous 1 × 10⁶ pre-irradiated ESCs vaccination three times at a 1-week interval, and 1 week after the third time of vaccination, 1 × 10⁶ LLCs were inoculated subcutaneously. Mice in the treatment groups 1 and 2 also received subcutaneous 1 × 10⁶ pre-irradiated ESCs immunization three times at a 1-week interval, at 2 and 5 days after the tumor challenge, respectively. b After the inoculation of 1 × 10⁶ LLCs, tumor size of all groups was measured using a digital caliper and tumor volumes were calculated. The results indicated statistical difference in tumor size between the vaccine and control group from day 5 on (p < 0.05, and p < 0.01 from day 7 on), between the treatment groups and control group (p < 0.05) from day 7 on, and between the treatment groups 1 and 2 from day 9 on. c At the end of the experiment, all mice were killed under anesthesia and tumors were excised and weighed. The results of tumor weight suggested that there was significant difference between the vaccine and control group (p < 0.01), between the treatment groups and control group (p < 0.05), and between the treatment groups 1 and 2 (p < 0.05). d Tumor formation time of the control group and treatment groups. The difference in tumor formation time between the control group and treatment group 1 was highly significant statistically (p < 0.01). Error bars denote SD

Fig. 2

The classification of lymphocytes in circulation. a Administration of ESCs induced significant proliferation of CD4+ and CD8+ lymphocytes in peripheral circulation of mice from the vaccine and treatment groups compared with the control group (p < 0.05), however, there was an obvious decrease of CD19+ lymphocytes (p < 0.05). No significant difference was detected among the vaccine and treatment groups. b The ratio of CD4+ and CD8+ lymphocytes of the vaccine and treatment groups become smaller (p < 0.05), so there were more CD8+ lymphocytes (cytotoxic T lymphocytes) generated compared with CD4+ lymphocytes

Fig. 3

The proliferation of lymphocytes in spleens of mice from all groups (a = control group, b = vaccine group, c = treatment group 1, d = treatment group 2). a The immunohistochemistry staining of CD4+ lymphocytes. The MOD (a = 0.14 ± 0.029, b = 0.25 ± 0.048, c = 0.19 ± 0.038, d = 0.23 ± 0.028) indicated significant difference of the vaccine and treatment groups compared with the control group (p < 0.05). b The immunohistochemistry staining of CD8+ lymphocytes. There was significant difference of MOD (a = 0.14 ± 0.032, b = 0.27 ± 0.044, c = 0.20 ± 0.047, d = 0.19 ± 0.034) of the vaccine and treatment groups compared with the control group (p < 0.05)

Fig. 4

Concentration of cytokines in serum of mice from all groups. Mice in both the vaccine and treatment groups had significantly increased expression of interleukin-2 and interferon γ (p < 0.05) compared to the control group. Moreover, mice with minor tumor load had increased expression in interleukin-2 (p = 0.102) and interferon γ (p < 0.05) compared with mice with heavy tumor load. No significant difference was found in concentration of IL-4 of all groups

Fig. 5

The cell apoptosis of tumor sections from all groups (a = control group, b = vaccine group, c = treatment group 1, d = treatment group 2). The MOD suggested that mice in the vaccine and treatment groups had more cell apoptosis compared with the control group (p < 0.05, a = 0.035 ± 0.0041, b = 0.051 ± 0.0052, c = 0.057 ± 0.0046, d = 0.050 ± 0.0040)