PD-1-siRNA delivered by attenuated Salmonella enhances the antimelanoma effect of pimozide

Abstract

Melanoma is one of the most aggressive skin cancers worldwide. Although there has been much effort toward improving treatment options over the past few years, there remains an urgent need for effective therapy. Immunotherapy combined with chemotherapy has shown great promise in clinical trials. Here, we studied the cooperative effects of the small molecule drug pimozide, which has a therapeutic effect in melanoma, and RNA interference (RNAi) targeting PD-1, an important immune checkpoint molecule involved in tumor immune escape. PD-1 siRNA was delivered by attenuated Salmonella to melanoma-bearing mice in combination with pimozide. Our results demonstrated that the combination therapy had the optimal therapeutic effect on melanoma. The mechanisms underlying the efficacy involved the induction of apoptosis and an enhanced immune response. This study suggests that immunotherapy based on PD-1 inhibition combined with anticancer drugs could be a promising clinical strategy for the treatment of melanoma.

Fig. 1. Construction and identification of the recombinant plasmids.

a Local secondary structure of programmed death 1 (PD-1) mRNA at the regions targeted by the three PD-1 small interfering RNAs (siRNAs) synthesized in the study. b Effect of three plasmids containing different sequences of PD-1-specific short hairpin RNA (shRNA) on EL4 cells for 24 h. c Quantification of PD-1 protein levels in (b) from three independent experiments. d Effect of three plasmids containing different sequences of PD-1-specific shRNA on EL4 cells for 48 h. e Quantification of PD-1 protein levels in (d) from three independent experiments. Media indicate untreated cells. The data are presented as the mean ± standard deviation (SD). **P < 0.01 vs the media group

Fig. 2. Attenuated Salmonella distribution in tumors and other organs.

At 24 h, 48 h, and 1 week after intraperitoneal (i.p.) injection of attenuated Salmonella, the mice were killed, and the tumors, liver, spleen, lungs, heart, and kidneys were resected, homogenized, diluted, and plated onto LB agar plates. a Representative images of LB agar plates. b Quantitative analyses of bacterial counts at 24 h, 48 h, and 1 week post injection. The data are presented as the mean ± SD of three separate experiments. **P < 0.01 vs the tumor group

Fig. 3. Antitumor effects of various treatments in vivo.

At day 14 after tumor implantation, the mice were treated with various treatments. a Analyses of programmed death 1 (PD-1), p-Stat5, and Stat5 expression using western blotting (WB). b Semiquantitative analyses of relative protein levels. c Images of representative tumors from every group. d Average tumor weight of each group. e Survival curves. f Tumor incidence curves for each group. ^aP < 0.05 vs the Mock or pSi-Scramble group. ^bP < 0.05 vs the pimozide group. ^cP < 0.05 vs the pSi-PD-1 group

Fig. 4. Effect of cotreatment on apoptosis in vivo.

a Apoptosis of tumor cells in each group detected by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end-labeling) staining. b Quantitative analysis of apoptotic cells in TUNEL assays. c Cleaved caspase 3 levels were examined by western blotting (WB) in B16 xenografts exposed to various treatments. d Semiquantitative analysis of relative protein levels. ^aP < 0.05 vs the Mock or pSi-Scramble group. ^bP < 0.05 vs the pimozide group. ^cP < 0.05 vs the pSi-PD-1 group

Fig. 5. Tumor-infiltrating T lymphocyte analyses.

Programmed death 1 (PD-1) expression (a) and the recruitment of CD4⁺ (b) and CD8⁺ (c) T lymphocytes in tumor tissues analyzed by immunohistochemistry (IHC). d CD4 and CD8 protein expression levels in tumor tissues detected using western blotting (WB). e Semiquantitative analysis of CD4 and CD8 levels in each group. ^aP < 0.05 vs the Mock or pSi-Scramble group. ^bP < 0.05 vs the pimozide group. ^cP < 0.05 vs the pSi-PD-1 group. The arrows point to IHC-positive cells

Fig. 6. The proportion of immune cells in the spleen after various treatments.

At 2 weeks after treatment, the spleen was excised, and the immune cells were evaluated using flow cytometry. Proportions of programmed death 1 (PD-1)-positive cells (a), NK1.1 cells (b), CD4⁺ and CD8⁺ T lymphocytes (c) and CD25⁺Foxp3⁺Treg cells (d). e The average percentages of CD4⁺ and CD8⁺ T lymphocytes, NK cells, and regulatory T cells (Tregs) were determined using statistical analysis. ^aP < 0.05 vs the Mock or pSi-Scramble group. ^bP < 0.05 vs the pimozide group. cP < 0.05 vs the pSi-PD-1 group

Fig. 7. Overview of the synergistic antimelanoma effect of pimozide and pSi-PD-1 in combination.

Pimozide kills tumor cells, thereby releasing more antigens and attracting more T cells to the tumor, and the addition of programmed death 1 (PD-1)-small interfering RNA (siRNA) interferes with PD-1 expression on the surface of T cells, thereby restoring the killing function of T cells and playing a synergistic antitumor role