Reactivation of the tumor suppressor PTEN by mRNA nanoparticles enhances antitumor immunity in preclinical models

Abstract

Increasing clinical evidence has demonstrated that the deletion or mutation of tumor suppressor genes such as the gene-encoding phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in cancer cells may correlate with an immunosuppressive tumor microenvironment (TME) and poor response or resistance to immune checkpoint blockade (ICB) therapy. It is largely unknown whether the restoration of functional PTEN may modulate the TME and improve the tumor's sensitivity to ICB therapy. Here, we demonstrate that mRNA delivery by polymeric nanoparticles can effectively induce expression of PTEN in Pten-mutated melanoma cells and Pten-null prostate cancer cells, which in turn induces autophagy and triggers cell death-associated immune activation via release of damage-associated molecular patterns. In vivo results illustrated that PTEN mRNA nanoparticles can reverse the immunosuppressive TME by promoting CD8⁺ T cell infiltration of the tumor tissue, enhancing the expression of proinflammatory cytokines, such as interleukin-12, tumor necrosis factor-α, and interferon-γ, and reducing regulatory T cells and myeloid-derived suppressor cells. The combination of PTEN mRNA nanoparticles with an immune checkpoint inhibitor, anti-programmed death-1 antibody, results in a highly potent antitumor effect in a subcutaneous model of Pten-mutated melanoma and an orthotopic model of Pten-null prostate cancer. Moreover, the combinatorial treatment elicits immunological memory in the Pten-null prostate cancer model.

Fig. 1.. Characterization of PTEN mRNA NPs (mPTEN@NPs) and expression of the tumor suppressor PTEN by treatment with mPTEN@NPs in vitro.

(A) TEM image of mPTEN@NPs. Scale bar, 100 nm. (B) The size distribution of mPTEN@NPs detected by DLS. (C) The size of mPTEN@NPs in PBS or medium did not change during evaluation over 24 hours. Data shown are representative of three independent experiments. (D) Cellular uptake of Cy5-mRNA@NPs (red) was characterized at different time points. (E) Cy5-mRNA@NPs (red) escape from endo/lysosomes (green). Nucleus were stained with DAPI (blue) before evaluated by confocal microscopy. (F) RT-PCR for PTEN mRNA in PTEN-Cap8 cells after indicated treatments for 48 hours. Cells without treatment (Ctrl) served as the background. Data shown are representative of three independent experiments. (G) Western blot analysis of PTEN expression in PTEN-Cap8 cells after the indicated treatments for 48 hours. Bottom: The quantitative analysis results for PTEN expression that were calculated by normalizing PTEN protein band intensity at each condition to that of β-actin by ImageJ software. (H) Immunofluorescence imaging of hemagglutinin (HA)–tagged PTEN (green) expression in B16F10 cells treated with naked PTEN mRNA, NPs, or mPTEN@NPs for 48 hours. Cells without treatment served as the control (Ctrl). Scale bars, 20 μm.

Fig. 2.. mPTEN@NP treatment induces ICD of cancer cells in vitro.

(A) Cell viability of PTEN-Cap8 and B16F10 cells after treatment with mPTEN@NPs for 48 hours. Data are presented as means ± SD (n = 3 replicates). Statistical significance was calculated using a two-tailed Student’s t test, ***P < 0.001. (B) Confocal laser scanning microscopy (CLSM) imaging for cells transfected with GFP-LC3. (C to E) Analysis of ICD markers in PTEN-Cap8 and B16F10 cells after mPTEN@NPs treatment for 48 hours. (C) CRT expression on PTEN-Cap8 and B16F10 cells was evaluated by CLSM. ATP release (D) and HMGB1 release (E) were evaluated by ELISA. (F) CRT expression was assessed by CLSM on PTEN-Cap8 and B16F10 cells that were cotreated with mPTEN@NPs and the autophagy inhibitor, 3-MA. (G) Western blot analysis for PTEN and LC3-II expressions when cotreated with mPTEN@NPs and 3-MA for 48 hours. Bottom: The quantitative analysis results for LC3-II expression that performed on LC3-II protein bands intensities at each condition were normalized with β-actin. (H) ATP release after treatment with mPTEN@NPs and 3-MA for 48 hours was evaluated by ELISA. Data in (D), (E), and (H) are presented as means ± SD (n = 3 replicates per group) and were calculated via one-way ANOVA with a Tukey post hoc test. **P < 0.01 and ***P < 0.001. Scale bars, 20 μm.

Fig. 3.. mPTEN@NPs induce antitumor immune responses in the Pten-mutated B16F10 tumor–bearing mouse model.

(A) Experimental timeline for treatment of B16F10 tumor–bearing mice. S.C., subcutaneous; iv, intravenous. (B) Tumor weights of B16F10 tumor–bearing mice treated with PTEN@NPs. Data are presented as means ± SD (n = 4 mice per group). (C to E) Flow cytometry analysis results of the percentage of CD11c⁺MHC-II⁺ LNDCs (C), and the percentage of CD3⁺CD8⁺ T cells (D) and IFN-γ⁺CD8⁺ T cells (E) isolated from the tumor. Data are presented as means ± SEM (n = 4 mice per group). (F) Immunofluorescence imaging from Pten-mutated B16F10 tumor tissues shows CD8⁺ T cell infiltration (green) after treatment with saline, NPs, or mPTEN@NPs. DAPI (blue) stains nuclei. Scale bars, 50 μm. (G and H) Flow cytometry analysis result of the percentage of Foxp3⁺CD25⁺CD4⁺ T cells (G) and Mo-MDSCs (H) gating on CD11b⁺Ly6C⁺Ly6G⁻ cells. Data are presented as means ± SEM (n = 4 mice per group). (I to K) ELISA analysis results of cytokine in the supernatant of excised tumors from mice (n = 3 mice per group), including TNF-α (I), IL-12p70 (J), and IL-10 (K). (L) Immunofluorescence imaging of PTEN (green) and LC3-II (red) expression in Pten-mutated B16F10 tumor tissues after the indicated treatments. (M) Analysis of ATP release in the supernatant of excised tumors from mice treated with saline, NPs, or mPTEN@NPs (n = 3 mice per group). Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.. mPTEN@NPs increase the therapeutic efficacy of anti–PD-1 in the Pten-mutated B16F10 tumor–bearing mouse model.

(A) Experimental timeline for treatment of B16F10 tumor–bearing mice. (B) Individual growth curves for mice treated as indicated. (C) The average tumor growth curves for mice treated as indicated. Data are represented as means ± SD (n = 7 mice per group). Statistical significance was calculated in (C) using a one-way ANOVA with a Tukey post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. (D) Immunofluorescence imaging of tumor tissues showing expression of HA-PTEN (green) and LC3-II (red) after treatment with mPTEN@NPs with or without anti–PD-1. (E) Immunofluorescence staining of tumors for CD8⁺ T cell infiltration (green) after treatment with saline, NPs, or mPTEN@NPs. (F) Immunofluorescence staining of tumors for CRT expression (red) after indicated treatments. (G and H) ELISA analysis of ICD markers HMGB1 (G) and ATP (H) in the supernatant of tumors excised from mice treated as indicated. Data are presented as means ± SD (n = 3 mice per group). Statistical significance was calculated in (G) and (H) via one-way ANOVA with a Tukey post hoc test. *P < 0.05 and **P < 0.01. Scale bars, 50 μm.

Fig. 5.. Therapeutic efficacy and immunological memory of mPTEN@NPs with anti–PD-1 in the orthotopic mouse model of Pten-null prostate cancer.

(A) Experimental timeline for treatment of Pten-null orthotopic prostate tumor–bearing mice. (B) In vivo bioluminescence imaging of Pten-null orthotopic prostate tumors from mice treated as indicated. Tumor imaging was obtained every 5 days from the initial treatment day (day 10 after tumor inoculation) until day 25. (C) The fold change in bioluminescence signals from baseline at day 10 of PTEN-Cap8-Luc tumors. (D) Quantitative analysis of the fold change in bioluminescence signals from baseline of PTEN-Cap8-Luc tumors at day 25. Data in (C) and (D) are presented as means ± SD (n = 3 mice per group). Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test. *P < 0.05. (E) Immunofluorescence staining of tumors for PTEN (red) and LC3-II (green) expression at day 25 after the indicated treatments. Scale bar, 100 μm. (F) Immunofluorescence staining of tumors for CD8⁺ T cell infiltration (green) at day 25 after the indicated treatments. Scale bar, 100 μm. (G) Quantitative analysis of mean fluorescent intensity from (F). Data are presented as means ± SD (n = 3 mice per group). Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test. ***P < 0.001. (H) Experimental timeline for treatment of Pten-null orthotopic prostate tumor–bearing mice and S.C. rechallenge. In the naive group, C57BL/6 mice were subcutaneously implanted with PTEN-Cap8 cells at day 0 without any pretreatment. (I) Representative photograph of mice from the naive group and the combination treatment group at day 25. Note that the mice in the naive group are about 7 to 8 weeks younger than those in the combination treatment group. (J) The subcutaneous tumor growth profile for the naive group and the combination treatment group (mPTEN@NPs + anti–PD-1). Data are presented as means ± SEM (n = 4 mice per group).