Combined Tumor Cell-Based Vaccination and Interleukin-12 Gene Therapy Polarizes the Tumor Microenvironment in Mice

Abstract

Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression. Combining immunotherapy with antiangiogenic/antivascular therapy might be an effective therapeutic approach. The aim of our study was to elaborate an anticancer therapeutic strategy based on the induction of immune response which leads to polarization of tumor milieu. To achieve this, we developed a tumor cell-based vaccine. CAMEL peptide was used as a B16-F10 cell death-inducing agent. The lysates were used as a vaccine to immunize mice bearing B16-F10 melanoma tumors. To further improve the therapeutic effect of the vaccine, we combined it with interleukin (IL)-12 gene therapy. IL-12, a cytokine with antiangiogenic properties, activates nonspecific and specific immune responses. We observed that combined therapy is significantly more effective (as compared with monotherapies) in inhibiting tumor growth. Furthermore, the tested combination polarizes the tumor microenvironment, which results in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch manifests itself as a decreased number of tumor blood vessels, increased levels of tumor-infiltrating CD4(+), CD8(+) and NK cells, as well as lower level of suppressor lymphocytes (Treg). Our results suggest that polarizing tumor milieu by such combined therapy does inhibit tumor growth and seems to be a promising therapeutic strategy.

Keywords: CAMEL; Combined anti-tumor therapy; IL-12; Polarization of tumor microenvironment; Tumor cell-based vaccine.

Fig. 1

Tumor cell-based vaccine construction. Induction of necrosis by CAMEL. a B16-F10 cells were treated with FITC-CAMEL, and then propidium iodide (PI) was added. Lens magnification: ×20 and ×63. The peptide (green fluorescence) localizes in the cytoplasm. With time, the accumulation of CAMEL in cells causes cell swelling and rupture of the plasma membrane, triggering cell death (red fluorescence). b B16-F10 cells treated with CAMEL (5–40 µM). Twenty-four hours later cells were stained with annexin V and 7-AAD and analyzed by flow cytometry. After treatment with 40 µM CAMEL >97 % cells were necrotic (annexin V⁺7-AAD⁺)

Fig. 2

Inhibition of B16-F10 tumor growth in response to combination therapy involving tumor cell-based vaccine and IL-12. a, b Seven days after inoculation (lower flank) with B16-F10 melanoma cells (3 × 10⁴/100 μL PBS⁻; n = 9) subcutaneous injections of lysate from CAMEL-treated tumor cells (1.8 × 10⁵/100 μL PBS⁻) were started (contralateral flank). The vaccine was administered three times, 1 week apart. In combined therapy regimen, 24 h after each vaccine administration plasmid DNA encoding IL-12 gene was additionally injected at the same site (50 μg/100 μL PBS⁻). c Combined therapy was highly effective in inhibiting tumor growth compared to controls receiving single-agent therapy. Compared to control, statistical differences on the 25th day of therapy were *P  < 0.01, **P < 0.05. d Photographs taken on the 28th day of the experiment. e Tumors (n = 3) were collected 4 weeks after challenge and counterstained with hematoxylin/eosin. Considerable necrotic areas (red arrows) and immune cell infiltration (blue) seen in tumor sections from mice treated with tumor cell-based vaccine, IL-12, or their combination. Magnification: ×20

Fig. 3

Induction of immune response by treatment with tumor cell-based vaccine and IL-12. One week after final drug injection, mice (n = 8) were killed. Cervical lymph nodes and tumor material were collected for flow cytometric analysis to determine the levels of T lymphocytes and NK cells. a Higher level of T cells in draining lymph nodes were noted in the case of combined therapy. Compared to controls, statistical differences were *P < 0.05, **P < 0.0025, ***P < 0.0005. b Significantly higher levels of tumor-infiltrating CD4⁺, CD8⁺ T cells and NK cells were found after combined therapy. Compared to controls, statistical differences were *P < 0.025, **P < 0.035, ***P < 0.02

Fig. 4

Reduced number of tumor blood vessels following combined therapy. One week after the last drug injection mice were killed, and tumors excised, fixed and stained with antibody against CD31 (marker of endothelial cells, green fluorescence). The number of vessels was counted for each experimental group in ten visual fields from four tumor sections (magnification: ×20). Significantly decreased numbers of vessels were found in tumor sections from mice treated with combined therapy as compared to controls. Compared to control, the statistical difference was *P < 0.0001

Fig. 5

Reduced level of Treg lymphocytes after combined therapy. On the 28th day of the experiment, tumors (n = 8) were excised. Single-cell suspensions obtained were then used to quantitate Treg lymphocyte levels. The percentage of Foxp3⁺CD25^high+ regulatory lymphocytes (subpopulation of CD4⁺ T lymphocytes) was determined from the lymphocyte population gate. The largest decrease in the level of tumor Treg lymphocytes was found for the group of mice treated with combined therapy. Compared to controls, the statistical difference was *P < 0.03

Fig. 6

Polarization of tumor microenvironment by tumor cell-based vaccine and IL-12. Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression (allowing cancer cells’ escape from immune surveillance) (Hanahan and Coussens ; Szala et al. 2010). Combining immunotherapy with antiangiogenic therapy might be an effective therapeutic approach (Huang et al. ; Tartour et al. 2011). The combination tested seems to polarize the tumor microenvironment, resulting in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch appears as a decreased number of tumor blood vessels, increased levels of CD4⁺, CD8⁺ T cells and NK cells, as well as lower levels of suppressor lymphocytes (Tregs) in tumors of treated mice. Ultimately, this results in tumor growth arrest