Powerful anti-colon cancer effect of modified nanoparticle-mediated IL-15 immunogene therapy through activation of the host immune system

Abstract

Rationale: Colorectal cancer (CRC) is the third most commonly diagnosed cancer around the world. Over the past several years, immunotherapy has demonstrated considerable clinical benefit in CRC therapy, and the number of immunologic therapies for cancer treatment continues to climb each year. Interleukin-15 (IL15), a potent pro-inflammatory cytokine, has emerged as a candidate immunomodulator for the treatment of CRC. Methods: In this study, we developed a novel gene delivery system with a self-assembly method using DOTAP and MPEG-PLA (DMA) to carry pIL15, denoted as DMA-pIL15 which was used to treat tumor-bearing mice. Results: Supernatant from lymphocytes treated with supernatant derived from CT26 cells transfected with DMA-pIL15 inhibited the growth of CT26 cells and induced cell apoptosis in vitro. Treatment of tumor-bearing mice with DMA-pIL15 complex significantly inhibited tumor growth in both subcutaneous and peritoneal models in vivo by inhibiting angiogenesis, promoting apoptosis, and reducing proliferation through activation of the host immune system. Conclusion: The IL-15 plasmid and DMA complex showed promise for treating CRC clinically as an experimental new drug.

Keywords: DOTAP; MPEG-PLA; colorectal cancer; immunotherapy; interleukin-15.

Figure 1

Transfection and function of pIL15 in vitro and in vivo. MPEG-PLA and DOTAP were assembled into a new gene carrier—DOTAP/MPEG-PLA (DMA) micelles—which carry the pIL15 plasmid into cancer cells. Cancer cells then express and secrete IL15, which stimulates host immune cells and promotes proliferation and secretion of immunogenic factors that help kill tumor cells.

Figure 2

Interaction modes between polymer and DOTAP revealed by molecular dynamics simulations in a water environment and in an aqueous environment after running for 300 ps. (A) The initial conformation of polymer complexed with DOTAP. (B-E) Conformations corresponding to snapshots of the complex collected at 4.990 ps, 9.970 ps, 15.015 ps, and 300 ps, respectively. Polymer is represented with thick stick, while DOTAP is depicted with scaled ball and stick style. Two terminal heavy atoms in the polymer and the head heavy atom in the compound are highlighted using CPK style. (F) Polymer is represented with thick stick, while DOTAP is depicted with scaled ball and stick style. Two terminal heavy atoms in the polymer and the head heavy atom in the compound are highlighted using CPK style. (G) Polymer is described with a solid surface and the part corresponding to [C2H5O] is colored pink. DOTAP is represented in the same way and its carbon atoms are colored green.

Figure 3

Preparation and physicochemical properties of DMA. (A) Schematic depiction of DMA/pIL15. (B) Zeta-potential of DMA. (C) Morphological characteristics. (D) Particle size of DMA. (E) Gel retardation assay of DNA and complexes: lane 0, DNA marker; lanes 1 to 3, naked pIL15; lanes 4 to 6, N/P ratios of DMA/pIL15 (1.25:1); lanes 7 to 9, N/P ratios of DMA/pIL15 (2.5:1); lanes 10 to 12, N/P ratios of DMA/pIL15 (5:1). Results show that pIL15 is completely incorporated into DMA at a N/P ratio of 5: 1 and that complexes are prepared without free DNA.

Figure 4

Measurement of transfection efficiency in vitro and in vivo. DMA containing pEGFP (4 ug) was used to transfect CT26 cells at a weight ratio of 1:10 pEGFP to DMA. Transfection efficiency was measured by flow cytometry (A) at a N/P ratio of 5:1 after 24 h with serum-free medium and (B) at a N/P ratio of 20:1 after 24 h with 10% serum of transfection. (C) Detection of IL-15 in cell supernatant from different groups by ELISA (Mean ± SEM, n=3, *, p < 0.01, DMA/pIL15 versus GS, DMA, DMA/pc3.1). (D) Bio-imaging analysis showing that tumors of DMA and DMA/pcDNA-toto-3 can be visualized by fluorescence due to uptake of DMA containing pcDNA-toto-3.

Figure 5

Lymphocytes stimulated by the supernatant of transgenic tumor cells and cytotoxicity of lymphocytes supernatant. When CT26 cells were transfected with DMA, DMA/pc3.1, or DMA/pIL15, or treated with GS as a negative control, for 72 h, the supernatant from different treatments were used to culture lymphocytes over the course of 24 h. (A) Proliferation of lymphocytes was detected by CCK8. Secretion of IFN-γ and TNF-α in the lymphocyte supernatant was detected by ELISA (mean ± SEM; n=3; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). When CT26 cells were treated with GS or transfected with DMA, DMA/pc3.1, or DMA/pIL15 for 72 h, the supernatants from different treatments were used to culture lymphocytes for 24 h. Resulting supernatant was then used to culture CT26 cells for 24 h, and the activity of CT26 cells was assessed by using the CCK8 test (B) (mean ± SEM; n=5; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). Apoptosis of CT26 cells was also detected by flow cytometry 24 h after adding the supernatant of lymphocytes. Results from flow cytometry are shown for (C) untreated CT26 cells, (D) the GS group, (E) the DMA group, (F) the DMA/pc3.1 group, and (G) the DMA/pIL15 group.

Figure 6

Anti-tumor effect of DMA/pIL15 in the subcutaneous model of colon cancer. (A) Tumor growth curves (mean ± SEM; n=6; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (B) Tumor mass (mean ± SEM; n=6; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (C) Body weight of mice (mean ± SEM; n=6; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (D) Images of tumors.

Figure 7

Anti-tumor effect of DMA/pIL15 in the peritoneal model of colon cancer. (A) Body weight of mice (mean ± SEM; n=6; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (B) Tumor mass (mean ± SEM; n=6; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (C) Images of mice and tumors. (D) Volume of ascites (mean ± SEM; n=6; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (E) Number of tumor nodules (≥3 mm) in different groups (mean ± SEM; n=6; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (F) Number of tumor nodules (<3 mm) in different groups (mean ± SEM; n=6; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1).

Figure 8

Expression of IL-15, IFN-γ, TNF-α, and IL-12 in vivo. (A) Ascites were collected after the final treatment, and levels of IL-15 expression were measured in the four groups (GS, DMA, DMA/pc3.1, and DMA/pIL15) of the peritoneal model (mean ± SEM; n=3; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). Tumor tissue of the subcutaneous model and ascites of the peritoneal model (GS, DMA, DMA/pc3.1 and DMA/pIL15) were collected after the final treatment, and expression of (B) IFN-γ, (C) TNF-α, and (D) IL-12 were measured in the tumor tissue and ascites by ELISA. (For (B-D), mean ± SEM; n=3; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1).

Figure 9

Proliferation of lymphocytes and increase in the numbers of cytotoxic T cells in the peritoneal model. Splenic cells from the peritoneal model (GS, DMA, DMA/pc3.1, and DMA/pIL15) were collected after the final treatment. (A) Expression of IL-15 promoted the proliferation of CD8+ T lymphocytes and CD4+ T lymphocytes. (B) Splenic lymphocytes were tested against CT26 cells at different E:T ratios by a standard 4 h ⁵¹Cr release assay (mean ± SEM; n=3; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1).

Figure 10

Detection of cell proliferation, tumor angiogenesis, and cell apoptosis. (A) Cell proliferation was assessed by counting the number of Ki67-positive cells in the field, and less cell proliferation was observed in the DMA/pIL15 group compared to the other groups (mean ± SEM; five high power fields per slide; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (B) Angiogenesis was assessed by counting the number of CD31-positive vessels in the field, and reduced angiogenesis was observed in the DMA/pIL15 group compared to the other groups (mean ± SEM; five high power fields per slide; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1). (C) Cell apoptosis was assessed by counting the number of TUNEL-positive cells in the field, and a greater level of apoptosis was observed in the DMA/pIL15 group compared to the other groups (mean ± SEM; five high power fields per slide; *, p < 0.01 DMA/pIL15 versus GS, DMA, DMA/pc3.1).