Melanoma stem cell vaccine induces effective tumor immunity against melanoma

Abstract

Melanoma stem cells (MSCs)-based vaccine strategies have been a potent immunotherapeutic approach for melanoma treatment, which aimed at inducing specific anti-tumor immunity and targeting cancer stem-like cells. As the main cancer-fighting immune cells, CD8⁺T cells play an important role in vaccine-induced antitumor immunity. Here, we developed a novel MSC vaccine that induces CD8⁺T cells to target melanoma stem cells specifically. The MSC vaccine was prepared for our study in order to determine the effectiveness of antitumor immunity. The proportion and activity of CD8⁺T cells were examined in the spleen after immunization, in particular, the expression and cytotoxicity of the immune checkpoint of spleen lymphocytes were detected by flow cytometry and ELISA, moreover, tumor size and the number of lung metastasis nodules were observed and the specific killing effect of the vaccine was evaluated in immunized mice. We found that the MSC vaccine could promote DCs maturation, activate CD8⁺T cells, suppress the expression of CTLA-4, PD-1, and Tim-3, and increase the expression of IFN-γ and GzmB of CD8⁺T cells. Melanoma growth and metastasis were inhibited by the vaccine's specific targeted killing effect. The vaccines based on melanoma stem cells (MSCs) delay the progression of melanoma by inducing anti-tumor immune responses in CD8⁺T cells.

Keywords: CD8+T cells; Melanoma stem cell; immune checkpoints; melanoma; vaccine.

Figure 1.

MSC vaccine improves the expression of marker of mature DCs and levels of IFN-γ and TNF-α. (a) Bone marrow-derived DCs from C57/BL6 mice were treated with different groups for 4d. The expression of CD80, CD86 and MHC II is detected by flow cytometry. (b) Different groups were pretreated with DCs for 4d, and then co-cultured with spleen cells for 6d. The secretion of IFN-γ and TNF-α in supernatant was determined by ELISA. (n = 3; *P  < .05，**P  < .01，***P  < .001).

Figure 2.

MSC vaccine increases the number of CD8⁺T cells and activates them. (a, b) Splenocytes were isolated from different groups. The expression of CD3, CD8 and CD69 in splenic lymphocytes of mice in different groups was determined by flow cytometry. (c) Immunohistochemical analysis of CD8 in tumor tissues. (n = 3; *P  <  .05, **P  <  .01).

Figure 2.

(Continued).

Figure 3.

MSC vaccine decreases the expression of immune checkpoints and enhances central memory CD8⁺T cells. (a) Splenic lymphocytes were harvested in 3 weeks after the last immunization. Flow cytometry was used to determine the expression of PD-1, CTLA-4, Tim-3. (b) CD8⁺T cells were obtained 3 weeks after the last immunization. Intracellular IFN-γ and GzmB in CD8⁺T cells were examined by flow cytometry. (c) Serum was collected from eyeball blood of the mice. Levels of IL-2, IFN-α, TNF-α and IFN-γ were detected in serum from through ELISA. (d) the expression of CD44 and CD62 L on CD8+T cells in different groups was determined by flow cytometry. The central memory T cells (Tcm, CD8+ CD44+ CD62 L+) in vaccine group was significantly higher than those in B16F10 and PBS groups, (n = 3; *P < .05，**P < .01, ***P < .001). (n = 3; *P < .05，**P < .01，***P < .001).

Figure 3.

(Continued).

Figure 4.

(a) Tumor size is measured at 2 weeks, 3 weeks and 4 weeks after tumor inoculation, including the maximum and minimum diameter of tumor. (b) At 3, 4, and 5 weeks after the tumor cells are injected into the tail vein of mice, the lung tissues are taken out and the number of metastatic nodules is counted. (c) The HE staining of tumor tissue. (d, e) The expression of Ki-67 and caspase-3 in tumors removed from C57/BL6 mice were detected by immunohistochemistry respectively. (n = 3; *P < .05，**P < .01，***P < .001).

Figure 4.

(Continued).

Figure 5.

MSC vaccine enhances the specific cytotoxic T-lymphocyte effects. (a) Splenic lymphocytes of different groups mice were collected as effector cells, and B16F10 cells as target cells. (b) Splenic lymphocytes in Vac-IL-33 group were collected as effector cells, and B16F10 cells, B16F10-CD44-CD133 -and B16F10-CD44⁺CD133⁺ as target cells. (c) MSC vaccine kills specifically CD44⁺CD133⁺ melanoma cells. The expression of CD44 and CD133 in melanoma tumor from different groups was evaluated by flow cytometry(n = 3; *P < .05).

Figure 6.

Scheme for the antitumor immune mechanism of MSC vaccine.