An effective cytokine adjuvant vaccine induces autologous T-cell response against colon cancer in an animal model

Abstract

Background: Despite recent advances in early detection and improvements in chemotherapy for colon cancer, the patients still face poor prognosis of postoperative recurrence and metastasis, the median survival for patients with metastatic colorectal cancer is approximately 22-24 months. Some immunotherapeutic approaches had been attempted in colon cancer patients to significantly increase overall survival. A vaccine based approach has shown a novel direction for colon cancer prevention and therapy.

Methods: In this study, the experiments were designed including prevention and therapeutic stages in order to attain effect against tumor recurrence in clinical settings. The anti-tumor efficacy of a novel cytokine adjuvant vaccine that contained cytokines GM-CSF and IL-2 and inactivated colon CT26.WT whole cell antigen was evaluated in BALB/c mouse tumor models by measuring tumor growth post vaccination and the survival time of tumor-bearing mice, analyzing the expression and distribution of CD4, CD8, CD11c, CD80, CD86 and CD83 positive cells in control and treated mice by flow cytometry and immunochemistry. The tumor-specific cytotoxic T cells (CTL) were analyzed by tumor proliferation and the lactic dehydrogenates (LDH) release assays. IFN-γ, IL-2 and GM-CSF secretion in serum was assayed by ELISA.

Results: Our results suggested that cytokine adjuvant vaccine significantly inhibited tumor growth and extended the survival period at least 160d. It was found that the levels of CD8 + T and the tumor-specific cytotoxicity were significantly higher in prevention and treatment group vaccinated by cytokine adjuvant vaccine. CD8 + T cells play a key role in anti-tumor response.

Conclusions: The novel GM-CSF and IL-2 based adjuvant vaccine effectively activated autologous T-cell response and represented a promising immunotherapeutic approach for patients with colon cancer.

Keywords: CT26.WT; Colon cancer; Cytokine adjuvant; Immunotherapy; Tumor vaccine.

Fig. 1

Standardization of the tumor model and its tentative mode of action The animals were vaccinated for two times before injected tumor cells during pre-experimental stage. Panel (a), shows the tumor growth after day 0, 10 and 30, as a result of subcutaneous injection of CT26.WT tumor cells into the neck of BALB/c mice. Panel (b), depicts the efficacy of cytokine adjuvant vaccine, where each group with 5 mice was vaccinated twice at interval of 7 and 3 days, before subcutaneous injection of CT26.WT cells. Panel (c), shows the comparison between male and female mice in terms of tumor uniformity. Panel (d), represents the standardization of inactivated antigen for effective anti-tumor response. M stand for million, * stand for Optimization. Panel (e), represents the effect of combination application compared to single group by observing survival period, which each group with 15 mice was vaccinated twice before subcutaneous injection of CT26.WT cells. *denotes p < 0.05, ***denotes p < 0.001. Panel (f), The experiment timeline showing the times of vaccinated. In order to establishing the tumor recurrence model stimulating the clinical status, the experiment was divided into prevention stage and therapeutic stage. The Prevention group was vaccinated vaccine for two times in prevention stage, the Treatment group was vaccinated vaccine for four times include prevention stage and therapeutic stage, and the Tumor group vaccinated inactivated antigen for control. All groups were inoculated tumor cells at the same time. Tumor inoculation was the initial point (day 0)

Fig. 2

Preliminary effect of the cytokine adjuvant vaccine Panel (a), shows the pictures of mice with tumors at day 49 from three different groups. Panel (b), shows the pictures of tumors excised from each group of mice and subsequently these tumors were used to calculate the tumor inhibition rate. Panel (c), represents the comparison of tumor volume (left) and weight (right) between tumor, prevention and treatment group. Panel (d), depicts a survival curve analyzed with a log-rank test of Kaplan-Meier curves using mice from Tumor (control), Prevention and Treatment group, where 20 mice of each group were followed for a period of 160 d (Kaplan Meier, p < 0.001). ***denotes p < 0.001

Fig. 3

Variation of CD4+, CD8+ cell populations in peripheral lymphoid organs Panel A, depicts the CD4/CD8+ and CD8/CD3+ ratios as analyzed by flow cytometry analysis from the Lymph node, LN (a, b) Panel B, depicts the CD4/CD8+ and CD8/CD3+ ratios as analyzed by flow cytometry analysis from the Spleen, SP (c, d), isolated from Tumor (control) and Treatment group mice at an interval of 3–4 d. *denotes p < 0.05,**denotes p < 0.01, ***denotes p < 0.001

Fig. 4

Tumor specific CTL activity in peripheral lymphoid organs and cytokines secretion in the serum The peripheral immune organs (LN and SP) were collected (3 mice/per group), and CD3+ T cells were isolated. These cells were incubated with CT26.WT tumor cells. Panel (a), represent the LDH release induced by T cells isolated from LN (left) and SP (right) of tumor control or treatment group mice. Panel (b), represents the CT26.WT tumor cell proliferation as measured by MTT assay post incubation with effector T cells at an E: T ration of 50:1. Panel (c), represents the secretion of cytokines measured by ELISA kit from the serum of tumor control and treatment group mice. Data are represented as mean ± SD. *denotes p < 0.05,**denotes p < 0.01, ***denotes p < 0.001

Fig. 5

Distribution of CD4+, CD8+, CD11c+, CD80+ and CD86+ cell populations in Lymph node and tumor tissue Panel A, represents the expression of CD4 (a), CD8 (b) and CD11c (c) as analyzed by IHC staining of LN isolated at different time points from tumor control or treatment group mice. Panel B, represents the expression of CD80 (a) and CD86 (b) as analyzed by IHC staining of LN isolated at different time points from tumor control or treatment group mice. The expression of these molecules was calculated by IHS. Panel C, represents the HE and anti-CD4 and -CD8 IHC staining of tumor tissues collected from different groups. The results were observed in (40 × 10) horizon. Data are represented as mean ± SD. Bar = 50 μm. *denotes p < 0.05, **denotes p < 0.01, ***denotes p < 0.001